rEVELOPMENT, EVALUATION, AND
IMPLE~ENTATION
OF A NEW DISCUSSION TOPIC FOR HONORS 299:
RESTRICTICN
~RAGMENT
LENGTH
POLY~CRPHISMS
Honors 499
Honors Thesis
by
Melissa A. King
Thesis Director:
Dr. Jon R. Hendrix
Ball
State University
Muncie, IN
May, 1991
-
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21
Appendi;.;
E ••
:=::e<3:::!:~ng
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II
ist.
II
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-
Introduction
resear:hers have
foun~
t~at
generatEd in all
are~s
of
possibly
TD~ay!
~2yord
~ic'=gy
t~e
ha~
Human genet cs is one
Suc~
rem 21.rk :;!J 1 e pc<, ':: e ,
2mou~t
to manage it
neces~ary
int~
jivided
~f
of knowladge being
biolcqV was escalating at a rate
w,~dom
~een
the
+hese
(Hurd 1989)
nearly 20,000 fields,
with
fiel~s.
progre~s
ge~~~ics
in
has brought with
it
ty to make decisioGs
concernir~
the Lse
o~
~ncw'~dS2
(Me I
.f.. ,..',
,..
:.he f :[',::,1 d
of
'--'
amniocente::,:; and
-; :;. ;', 9
geneti(:,~
r-:um='n
er'p r':, r-: t i. - ':::I
I
ar:
,:,t:"~r..
j
tic:'n,
2:,,:,9 en
':::;e>::
t2c~rogies
br'O'J9'~t
that they have
F:ecent ad v .'In c ::::OS in
,1 er'r ::;?':,
hc:,'/e
is:,su:::::s such as
~~,ene
sf"1 e'=t:,or:,
t:-;,::'r'ap',
'!
i c :;- t c: ItJar-'j :, :-- .,::: c: c7:I', t er' ,::If pub 1 i c:
,3nd
bE' '3enet i. c:':::' 1 1 y
~nd
cf
Becaus~
,::<,n,:: hi, ,--';;::':, "-, i c ,::<,:
1
\!
Ii, t ':;:r'ate
i
£.
i~s
Wt".'!
Dr'~A
;~'D;
a1
impact on
;,',i"-F~
to
! ..
\nde>- ':~tan d
T!U,'::. !-
- '.J..?
.::..pp':
1·'::,"
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it
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e'
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:n
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t
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1 ~.:' " .:
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bioi Cig/
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is
':'Clme cri ti.cal
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:::r"·,jer
d:::-:::
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i sc.:;u.e:=.:
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1_ ';
:::
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the
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qenetic -echnologies
One
-L
the
C~
goal -
.~e:r
~~-
of
• 1-'-'
I~ep
tc
":::.1"?
st~dents
~pdated
,.
'.p
'las
r·,::···_
:nuc!-';
rj '·-··c:.'<"Jn
i n
--
.~
c
".
is:ues with which
E~
'-,- di"ic'e
;enetic
en~tneering
fT,:,. t t
ei'~S
can affect 7any
The
d:~::
~se
of a new
DNA
"",/e
....
(BSC':3
c.,U. ick
~
,-. ..c
peop~E
. . /;::·:·· 0pITJen
~ecombinant
~
dS
3nC
oth~
t.
techn:lu~s
anj
:",
~;:.,
'':':
\/
;..
techncl~~y
~
F~'-'
-
3.gm,?r:t
"
~..:"
..
.......
~.
\
,1
T ,",
+.
~,
- .-::
'c iI" 9
t.
-
4
1::,18tting
variatic~
~ith
t~~
~attern
~2Ge
t~is
of
he very
hu~an
in a
indlvidual~
~edigree
are inherited and
technolg; In genetic diagnosis is 1 imited, but
0FL? jiagnasis
use~u~
restrictiJn fragment.
resu~ting
i~
useful
If
J_
'-
...
~-. '
digesti~n
when a certain
with a particular
a IT'utat. iDi'i occurs in a ',;Jene, the
T+ we compare the restr!ction3ps
fro~
~he
1 ike any other trait
ir~age
pect.ec: ,
a~d
1 9~::3~3) •
(Oppenh21mer et a1
e>~
tiversi~y
However, differences in
enz;me.
rest~iction
of frag8erts observed in
Us~
of the great
in the ge00me of the human populatIon, no two peop19
caG be followed
will
8ecau~2
lMc:lnerr!F'Y 1989)"
~ith
parti=ul~
a
(fragments
enzyme)
of normal
thcJse
Qf characteristic length)
is
~lwa)s
present
This
o~
absent
~estriction
in
~hE
site is
termed a marker.
important consequences:
detec~i~g
the disea3e
~Lbseque~tlv;
-
j
it may offer a
diag~.~stic
at a prenatal
~ither
and i t may lead to isolation
responsible for the
The :::)Dt <':'f'it:. i 3.1
di~2ase
procedure for
stage or
~f
the
g9n~
_.eWln 1-790).
'_!.se of RF--;_F'
.j
i.:;.;gnc3 i s:::.Tr·ent 1 I
"-'eq'__ l i. -es
re~ponsible
point mutation that affects the
gene anc the
C'ch
: inkage
ana~/sis
disorders
approach
ccu~j
gene)
be
u~timately
(these disorders
as heart
di~ease
te diagnose
u3e~
:~use~
~nd
restriction Lragment 38
fou"-' •
sympt~ms
(Qppenhe mer et a1
Huntington's DIsease, DNA
f~ag~ents
determ~natior
?s tc whether
m~Ce
polygen~=
diabetes mell itU5.
By digesting DNA of indivijua1s
can be
suc~
by the interaction of more than
be~ore
-
I~
(those disorders causej by a single gene), but this
~isorjers
a~e
is currently more conducive to manager
a~
appe2r by using
:988) on chromosome
~is~
for developing
can
r
net
a~
ind~vid~al
is
aHE·::ted"
The potential
for genetIc Cicgnasis
evident i- the rapid srowth ir the use of
diagnostIc laboratories.
Tab 1 e l l
j.
U~lns
~~is
RFLP technc'ogy
technique
Due to the
tech r o1ogy,
~he
topic of
Honors 279 curricula.
-
this
yea~,
increa~e
an entire
RF~P
in
has
~~e
b~en
Origina11y, the
l~cture
~~
st:::.
genes that can be detected by mappins to neart; RFLPs
3.1. 1990).
IS
~se~ulness
of
incor~or2~~d
tDO~C
was
(Marge et
t~is
ir~o
t~e
~p~tiDned
was devoted to ?FLP techco1
~Jring
:~y.
supplement and reinforce concepts present2d in the lecture, a
:3 c:: ,:i e
d~.
-.' t~ .::; '::;: e _. C
~a:~~ng
t~
E~ u. ~:: .~
r~3~by
n
~~
~~
':;J en E .."';
!-i .;'::: t
!J -:;:: d E' ~.: (? ':= t
c·:? "
.=~. ~1
r- CJ 1TI (J c=:. CJ en.-3. 1
2_i..;;I~_~;.:.:
'=:... ______. ______._____.______.__~~L_.~.cU_~_____ ..
_______lx.:_·om F.:FL F'
close
... 1
~~n~-,
dr'::~lt
":: j-. -"", CI r',
J.'=
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e f-'
t. :
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e
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E = ':c.'::!E:?r-·>or1:.::-·-
l.C
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:.;
Lt"] C' IT! ,:~.
m2'tal
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t. Cln
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.:=-; ~:~ i ~~ e
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t ::::' ~. . ~. ~.:
_'A:.
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~yctcnic
~y~-~JP~'I
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k:: :jr"'2\-
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"::'1
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rE~
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.?u. I:(]<3..CJi:,,····
.l·t
ver'y c' ose
.ery clDse
1-5 m.3p units
10 map units
:0 map units
'/et~y ':: lose
c
.::,\
x
C
'.~~
1'-,
and El ainE
Johans~~
: •• J "
......
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1
o':::;e
·/ery c 1 eyo!,?
5 :TI (3. p U.:1 i i:. ":3
-,
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Mar: '; e, ~~r-' t t-,I l1"""Genet i;:s: HUiT;.3.r< e.~~..::- f":'ct_:2.
t:· \~
Ap p r--:::J:-: j ..1\<:1. t e
map un:L ts
: oc.,:;.t ~ Dn
_.... ______
;~.:.,j
RFLPs.
1
I
1a~ge
iTl2i.j::
- 7
un its
(II .~,::" t-'
(
.~
I_.l r-· 1_
t:.Y';;'C) •
t·, S
.-
7
case study for use as a discussion was jeve10ped a= my
~0nGr~
I I. rlc:v:::rs:, 2';'9
Ho~ors
The
methods
l~ts
integ~ates
29 Q biolo3j cours?
t~e
c'assrs0m
"",r.
9 E' ;', e
~h~ch
are net fOJnd
OL
my
~orGrs
The purpose c+ the
mak~~g
c~
their peers.
hi '=,
OF"
in many othsr
tic::
i.
deve~rpment
various teaching
the
pr~jec~.
discua5i~n
S'Y'
CJ;.J
i
t:-~l
fi
were: Oncogenes and
t~pic~
S:OJ~
~3i\/idifig
Before the
'
:~
'J
ion s .
~n
~~ovid~
,':;"-"ge c'!
"T"\-,
F?
a':;5
the Honors
l.r-·t.e!
grCJu.p 5
~_~.·T!.:~·ll
,~... 1 , O\.'J
"
es-;:· f
setting than
:n
~he
classrocm.
~here
discussion leaders are
The format
~f
p~omote
to
di~~U5si~n~
the
discussion, whi 1 e also
vary, depending on the topic.
of the three
The materials pres2nted
topi~s
s
t
were each
of
7': ~2
E!2.C hi.:::.
~e~eloped
':
1.
Di~11U.l
by~'
f f rO'·'er. "
Fferent people.
For
e~3mpl~,
The
j~3cussi~n
Theref Dt-'e, the
the materials on
potential
carcinogens to which
In contrast to that
neural
tube defects
prenatal
may have been
st~dents
the discussion
app~aac~,
The steps
so reen in·:,. •,-r:et 1-, od s
c~
prenatal
(~.. (;2.
c,l ':)OC
di2g~osis
5·:', "ff) 1:.2 :e.,
The discussion
-
abc-tien
issue may be
the right to
kno~.
m2te~ial
on
(NTDsl relay::: .nformation about NTDs and
diagnosis.
'3.n d v ar i ou s
t~e
on cancer then
~~olded
anc
~he
~?j
U
~~e
out~
1 t r-··a ~';C>_'.:, c!
ired
1
lead to intense
focus moved to
ot~2r
tcp:~s
-Because the pos:::i.ble scenar-:o-::;
imitless, C2se stLdies can al so
conversation
s~~dent
in the discussion
to "engage in the
the rols of prospective
t~
used to facll !tate
Case stucies
gr~ups.
p3rent or sib1 ing of an affected
This is the approach
Sace studies can
~e
~sed
the
thou]ht process by playing
~cien~ific
~are~t,
21lo~
to
pro~cte
ta~en
in the
increased levels of
Any case
--
0+
s-tL'_dy cc:-:cern:'.'1g the impc-_ct
tee'-::--:log',' or, ti-1ei:-- 1 :L ... -es"
.r. ~
I
n
Th~
s_ r:ew tec:hnCl'rYTy' s!-:oul d
i:::::ol e,T---n
~
Th~s
989)
"force the
was our goal
RFLP Discussion
In order to Increase the
diagnosis and
intrc~uction
its importance,
to
~FLP
stude~t/s
~lrst
!
und2~s~anding
~egan
~ork
of RFLP
or an
techn01ogy.
appl ies to genetic dia9nosis.
The goal
was to take the compleN
in text
bco~s
and present
it
in such a way that nonbio'cgy
students would be able to understand anc
~0preciate
the
-
1"
In·!.C·:·-·Ti~~."':ic:.';'! •
--. ~~, .~.:!
.3.p P 1 '/
-:.0
-+- !-.
.
i s·
:;:,
:-~ ~'-.
:: _'.t,.
e c:. t.
i+e
1- ~ ..
'-:
..
of
ap~::'
ie-at ion of
The -- -" .. c
~~
~.D
~~,
"
only a- -, ios
t~~
te~~no~~gy
the
to a
_ the
~se
~f
s~ch
.1.1 i
t. I::.) c~! -,
there are
:-:~ght
et~i~31
to pri'i3cy! t ;-, .:. ~.
in i t i a 1
j I..!dgement
c:jj
c
::. .::~ -:;:. Lt e :~.
.-,,-.-i-
, •..J '._
t
t ::'::"
.' ,::;,c, CO::
!:;'f:-;
D
t ,,,.:: ;-::-.,::: '!
'he
.....
..}
, i.-:::"
"-
CJg .'
Cj:/ l
the L',:::··:':\
_ .-..
Cfl2.y
.-
11
:ormulate a situation
~./he
~or
questions
rrovi~~
~;t'tch/,
the C.,:'.se
group
was
3
~o
proceed
su]gested
wit~
Aiong
oase 3tudy,
commercial
~
in
~~ny
re~dins
1
also co
0cted
services
~vailable,
riC
1 uded
infor~2tion
-
~~?
This was intended to
r~l
so provided
(see Appendlx E).
Cli:::ta:l ne
concerning the
Ft-'om Gene 9,::reen, a
i. nf ormat i Dn a.bout
F:FLF'
ded me a fc'jer and pamphlet with
211
The information inclujed the types of
the cost of
tes~~ng,
testi~g
under
and a pamphlet eplaining the use of RFLP "
:~
n the pack et i s
~
r"if c~- '",at ion obt a i ne::J f
The~
en
1 ist of ·=;uggested
testing.
patern:~y
i
2.
e:planatory information and the
..:.i . .:::-, ,
They pre.
stu~e~ts.
~eVelD)ment.
E:>~
feel
discussion, while also allowing
aspects of RFLP rechnology.
serv::es.
?99
compi.' ed
different directions.
intro-~ctorv
the
I
~Dnors
~le
~
woul
s/he would make.
ist resarding RFLP
~I··
r21at?~
:
~/he
what
th8 dis=ussion group leader.
a b2Sis for begln0:ng
~he
imag~~e
decisi~ns
would 2Ct, what
a.~.::comp2.~ly
To
an Honors 299 student coult
~eascnably
inject him/herself and
ike, how
whic~
into
kinds of testing
Appendi~
discusston questions
f~r
also
a~~ilab'e
r
cOGtain~
proyi:~t
r~cm
I n'~
me with
2nd the costs
~?S1Tatecj
informa~lon
"~volv2d
with
the RFLP p2cket th.t is
the discussion sroGp leaders.
-
12
IV.
Evaluations
discussion was implemented
c~
~
t-ial
basis
~ith
three
d~scussion
This '1lat'?rial
~ere
completed by bsth the
199 (Honors 299)
s~ud9nts.
d:scussiJ~
~2
1.S
group leaders and the Bio
evaluation questions used and the
Overa 1 1 , the
Table
~
gives
3n
overview of the
T E. >},L~_.~~_____________ ..... _ .__ ._. ___ ...._...___._______._________ ........ _....._........ ___._ _ _ _ _ _
aspect o{ material
~~
f~.
T
!--''2'~3por: SES
clarity, understandabil ity
comprehendabil ity
-
9Li • 1
questicns
.----------.._ - - - _ ......_----_...._----
o
-
12
However, the evaluation also
too~
I
an~
im~le~ented
t~e
Two of
i~portan+
most
ac~~mpl
~ues~tnnc
the
attemptr:.'d to m'-'f:e t.r;e
~'::J 1
SuSg~stions
i0to accourt
sUJgesti~ns
st~dents'
I
ql.est:.cns.
2~d
study
for
~hes~
qlor~
0
suggestions
~t
fi'L:'!
1 ives and add more variety to the
th~s
'shed
~he
were: make the material
end
by altering
case
I
also
j::·.nfC)f"Tation mm-e under·::;;.tandabl e
t~2se
with
t~e
the case study.
of
1 m'i i r;::!:,:·ome .::.fU";e <::;'!_"]gest i on'::; "ade hy the
1 eacers
f2r
those that were feasijle in the second revision.
more pertinent to the
ethical
as~ej
c~anses,
c~
i. scuss i. on
I also compiled
3
b'y'
9 ~-·ou.p
more
Included in this 1 lst are
~rticles
0n PFLP
0fter making
teste~
again
~esporses
t~e
~uring
wer~
ted
~el
~cpics
use
i.'~'..c
'~0dents
expressed an
appropriate changps, tre materi:1
the
~al
~emester
of
was
the~
1990.
very positive.
Tacle 3, and the det311ed
fut'-'.'~f?
the
~hat
respc~;es
3re
3vailc~le
jn
;::: c'n d i
~<
D.
thi~::.-,atef"·ial
The
fln~~
~roduct
W3S
i.~cludec
as one of
~~2
}f
1991.
The
decisi~n
to include thic
matE~ial
In place
~~
ene
-
1.4
of the other' e}:'isting d:>::;CLlsi':;on tc:.pic:: ,,·)a·;; ma.de by Dr. "!endri:'
Tab 1 ~_~. ___._____________._.__ .____ .___________ ._
aspect of
!. of responses
ma~erial
_________ ~c..:?__:l.:.J..;:'"'_·J"_··
P''''.
____________
C)-~
-
Wr·~:.
1: ter-'
,-(:.::~t. .
,,-:1
er i a;
'!
-'-'nc.: E==-:901-': Ca:. .:t=. ,1: . ·..:.v..::e=--_
::.2
n
.~
2.2
u~derstanjing
8.nd
o~
techno'o;;
E'.pp1lcE,t.ic;r.'O:.
9.1
7
v-~C[-
c:.·
...:.:..
. .-,
...:.:..
4.':"
__ _------------------ ._--------_._---..
RFLP Laboratory
~s
--
•
thwh i 1 e
._------_._--------_._---_......
V.
,...,
,-,
,-;--:'
a final
labo~atory
addi~:cn
exercise was
laboratory material
tha~
to the
Ho~~~s
im~lemented.
has
bee~
2q 9
c~rriCLlla,
pendix
~
~n
RF~P
cont~~ns
incarporatet irto
Hon0r~
the
299.
deal irg with RFLP
none of the lab
Honers 299
tec~nDlogy
exerc-~es
~~j
its
~ubl is~ed
~)pl
wer~
ication and found tha
suitatle for use in the
~~~ri=ula,
~'Jel~e
Duri:';g t:-,e tii"Tl,:':; ItJe
Hencr i :'< found a
l,:;i::
e:~
er:::::. ':;e
U';
;::;
d~:"iel
Dp'lr-;,] th:,s : ab, Dr.
1 .=.\boratory manual
2:1t it 1 ed
The exercise is a
cl ip
weIr""'\-::
lab that very
We chDse
I::'
t~
paper
portrays hOW RFLP technology
effe~tively
ef~orts
abandon our
at developing a new lab
This
,
~"J
Afte~
gairing the
begar preparation for
the
exe~ci~e
ga~~ored
•
,
r~3hts
a.id t;-·e s·l- dents in understanding
use
t~
lts use.
the necessar;
Dr,
~aterials.
in the past.
the technologv works, and
-
.L
the RFLP discuss.or.
~t
will
thi~
"aboratory exercise, we
Hendri~
and found a few errors.
that had been shown
~ith
1
~"
wh~ch
The
and
I each practiced
we corrected,
exprc~se
was
The lab matorial
be used
~n
and we
implemented
was
fi~st
the future alcng
used
-
16
VI.
':Dncl U':;iDn
The RFLP discussion
~onors
aquaint the
technolog~
t~e
and
The RFLP material
~~cluding
~99
mate~ial
was developed
order to
students with a rew r2combinant
et~ica~
issues
cortai~s
factual
i~form3tion
i~
G~
inh~r2nt
D~A
i , such techniques.
infDrmation about RFLP,
screening, the history and development
The case
study explores the ethic21
issues resulting from the appl ication
of RFLP technology to gEGeti= diagnosis.
-
usirJ this discussion
understanding
i
~sues
are to enhance student
RFLP technolo9Y, provoke thDught about ethical
~f
i nher'el-I+,
materia~
The primary goals when
i~:
such t"'c'cr-:no 1 oc.~,/. ,::.nd :!-'pro"/e dec is ion-mal:: i ng
sk .i " 13 •
From the evaluatiDns,
it
:3
apparent that these goals have
Th2 students report
they better
unt~rstand
RFLP
th~t
technol~g,/
after the discussion
end most students were
According to group leader E/aluations as we'l
student
~-\jDrthwh
-
evaluatio~s,
the
d~scu~siDns
went ver
as Honors
wel-
~99
and the were
i 1 e,
accumulates
discussion
al~ost
tD~ics
dal~v.
wit~
HCJ!lors, 299 a:--=? kept
~e~
ab~-ea5t
8y
alt~ring
or replacing preV!DUS
information, the students e,rDl1ed ln
c)f ne',-J tec::ilnol':'3ies that may affec:'::.
17
theit~
1 ives.
Honors 299 curricu10 for regu12r usa
IS
one
~f
severa
Th's process of updating must
c:ontliluC;L=:.,
a.i1d
tj-·:e ::-;::-i...F' di.scu=".tOil mu.st
replacet with 'iew lnforT2tion.
-
b~
someday be dpdated or
-
~.
~~OS5~RY
•
JF TERMS
8
.9
analysis.
'I~ process of ide~t!fyi~~ the conceptual com;onents
of a proble~, st2teme~t c~ issue~
I~ ethics, ana'ysis
concerns t.'1e reason'~-:; -:::'h?t~::'mE'Gn? ;..:,ffers tc ju·:;tify a /'""'u1 e
or 'I i. "..·tue.
The study and/or
bioethics.
~~pl ic~ticn
of ethical/moral
:;:::1 es to iS3ues ard prob1ems arising within the
c :J n -.: e< t '0 f the 1 ife scj~nces.
pt~'Lnc
conflict.
Occurs in ~thics when different justifications
contrsdictory :oncluslcns about what ought to be
or nc~ done.
pr~duce
dc~e
DNA.
Deoxvribonuc'elC acid; a molecu1e consisting of anti2arallel str?nds of polynuc120tides that is the primary
carrier of genetic informatior
ethical analysis.
A)p'yins 2t~ical principles and virtues to
actual situations and c~ses 1~ the precess of justification
ethics.
Th2 :;tudy of mar.ll lty.
genetic screening.
The U5~ OS genetic ~i3g~osis to detect
aff'2cted irldiv:.dual::; 2.nd ccH"riet~~; in a popu] ation.
linkage.
t~p
A condition in w~ich two or
same chromosome.
~cre
genes are found on
morality.
ConcE~ned with right and wrong ~:tion, and alsn with
character (whether we a~~ 30~d or jad geople).
moral
values.
wrong action (oughts) 3nd good
as distinguished f~om C.lt h er ve.'
mutation.
Any
c~ange
i~
t~E
an~
i .. :E'1S
~a~
•
sequence of
geG~mic
2NA.
ought(s)"
Th,:,. t. which ;!e are -:0 d'::l beC3use it 1-:::, t'-'e r~ight
to do; used to express d~ty Dr mora 1 obl ig~tion.
~.hing
prenatal diagnosis.
The detection cf a gertic jiseasp before
birth using such techniques 35 u1trasounj, amniocentesis
and C')S.
principles.
General guide~ ines to action that justify the more
soeciflc action guides or ~ules.
.20
restriction enzymes.
Enzyme~ tha~
,:: ec~ uen c??s· of DNA , . \ s.c.ta" 1 '-,' '::; i >~
~-l e.3.ve ~he DN(~.
short specific
in length,
recogniz~
c~
eiJht
~2ses
restriction fragment length polymorphisms.
RFLPs;
segments of
DNA resu~ting f~G~ j~gestior b' restriction enzymes that
v,:;..r·y i", length .::;.,-;,j ,::,':';n :::;:" (j i st:i "1gU i ·,"hf?d:me f rO;'il another
tht~·::
"::;11 ·,-::.e;:.al'"·Et~.:.D'·;
Sou t :", e~"", b 1 :It ~ i r", '~':<;
~3shi0~
':e~=hnlqU(?~:
sLl,::h
EtS
:::.:=L.. "='c::. '3.r-·e i r-l c'r i t
and are u~ed as m~~~ers for
e:
ectl'~ophorj~si'=
a.:,d
cae O;T: i nan t
s~ecific chromosomes or
'.?::~
ill
"-'I
'::;en?<:: •
val Lles.
-
St'<ncards
are
J~dged
to be worthwhile
'"'I
::.. !.
RF~P
-
~ATER!AL,
FALL 1990
22
-
The Use of RFLP in Genetic DiagnosIs
Currently there are several methods employed to detect carriers of
specific genetic diseases.
Some of the more common techniques include:
pedigree analys.is, biochemical testing, and direct clinical obsertJation
(Stine 437).
Recent
a~vancement
in recombinant DNA technology has led
to a ne''\/ mode of detection: RFLP, restriction fr'agment length
polymorphism.
In this ne''\/ technology, DNA probes, DNA str'ands that
r'ecognize their complementary DNA :.equence, .3.re used to detect the
presence of abnormal genes.
RFLP technology differs from other gene screening technologies in
that it cannot be applied to a population unless the disease-causing
gene loci is Known along ",lith the specific DNA s.equence of that gene.
Individuals can be tested, but populations usually cannot be screened
using RFLP tecnnology.
This is due to the fact tha" much genetic
information is needed from the indi'.Jidual·"s family members, especial!>'
the parents, in order to diagnose a patient.
Tests using RFLP based
diagnosis which could be used to screen a large population for a certain
disease .,..jill not be atJailable until the actua.l gene3.ssociated
disease is identified and cloned (Stine 438).
1,~ith
a
The work being done on
the Genome Pr·o.ject may maKe available such infor'mation, leading to the
use of RFLP
technology on a widespread basis.
The individual ized
nature of RFLP technologies embodies many ethical issues.
[,Jould your
parents or other relatives be ,,\/illing to release medical information and
donate tissue in order for RFLP based diagnosis to be done for you?
-
Ought they?
Ought they be required by law to do so?
A DNA polymorphism is defined as "any DNA variant recognizable by a
change in DNA sequence that occurs with a frequency of greater than one
23
percent" (Stine 234).
These polymorphisms, or variants, can be detected
by restriction enzymes that recognize and cut DNA at particular
sequences.
The fragments resulting from such a cut are called
restriction fragment length polymorphisms, or RFLPs (pronounced
r·iflips).
Each restriction enzyme cuts the DNA at a specific
nitrogenous base sequence; therefore, each restriction enzyme will cut
the DNA into fr'agments of characteristic lengths.
enzyme that recognizes the sequence GATTAG
~lJill
For example, an
cut a strand of DNA each
time that sequence occurs.
A difference in the DNA seql.Jence,
.Ihich may be a mutation, may
l!.
change the cutting site and is thus recognizable via RFLP.
mutation has occurred, the base sequence is altered.
-
occurrs at a particular cutting
Sl
If a
If this alteration
te, the recognizable sequence does not
o ccur' and the DNA i s t h usn 0 t cut (s tin e 234).
8 y ': Dm p a I" i ng the
"normal" DNA strand.'s fragments to the fragments of a DNA strand IJJi th a
mutation, differences can be recognized.
Researchers currently compare
DNA fragments of an indit)idual 1,lJith the DNA fragments of his or her
parents (and maybe other relatives as wel 1) to locate the site of a
mutation.
Once a map of the human genome is complete, or once the
actual gene for a disease is identified and cloned, researchers wi 11 be
able to compare the fragments of an individual to this information
without the need to involve other fami 1y members.
The presence of restriction fragment length po1ymorphisms (RFLPs)
aid in the detection of a number of inherited diseases (Defesche 55).
Recogn i z ab 1e DNA polymorph isms, or' marker DNA, are some time s ca 11 ed RFLP
-
alleles.
These RFLP "alleles" do not have a
are inherited in a Mendel ian manner.
recognizab~e
function, but
If a polymorphism cosegregates
24
--
(s~gregates
tog~ther
g~ne,
instead of independently) with a defective
then the hoJo are 1 inKed on the same stretch of DNA.
If the htjo are
1 j n ked, the pol ym 0 l' phi sm may h e 1p "f 1a gilt h e pr' e sen ceo f the de f ec ti'.} e
gene.
Thus there is the potential for diagnosis using RFLPs, even
though the exact location or structure (sequence) of the defective gene
may not be KnollJn.
II
Gene tic
marKers ser(JE' as 1andmarKs for the hidden genes in
diagnostic testing" (Stine 438).
Using RFLPs, scientists may be able to
determine on l,oJhich chromosome a defecti()e gene appears.
For example,
the Hun t i ngton s Gene appears on chr'omosome four in humans, as
I
determined by RFLP technology.
RFLPs can also be used to define a
region on a particular chromosome thus
-
narro~\jing
the effort to isolate .;..
particular gene.
The ultimate goal of RFLP use is to isolate the gene, to understand
for what product it codes, and to decipher how its
disease (Stine 234).
may cause
By mid-1987, more than one thousand human RFLPs
were available for research on gene mapping.
impact!
~alfunction
This has great commercial
Currently more than fifty companies ar'e using, or are planning
to use, gene probes to locate defective human genes (Stine 235).
information
IS
also being put to use in the construction of detai led
genetic maps of the human genome.
Eventually, every defective gene in
the human genome may be recognized by RFLP 1 inKage!
•
-
This
--
2:5
TabJe
I.
ExamoJe
DNA
tragments with restriction sites.
A.
A DNA fragment ~loIith tl,IoIO restriction sites.
8.
A DNA fr~gment INith onb one restriction
occurred at the
s~cond
site; a mutation has
site.
(
C.
A DNA
fragment with only one restriction site; a mutation has
occurred at the first site.
"'~
,
\
(0.
/' I
/
~/
A DNA fragment with no restriction sites; mutations have occurred at
both sites.
-\J
26
.-
Table II. Gene Screen: RFLP Related Services
TESTING OFFERED
Genetic Series
Alpha 1- Antitrypsin Deficiency
Cystic Fibrosis
Duchenne Muscular Dystrophy
HLA B27
Sickle-Cell Ane mia
Type I Diabetes Mellitus
Identity Series
Forensic Testing
Paternity Testing
TESTING UNDER DEVELOPMENT
Cardiac Risk Panel
Bone Marrow Graft Monitoring
Leukemia and Lymphoma Diagnostics
Neurofibromatosis
Prognostic Test for Breast Cancer
(
OTHER SERVICES AVAILABLE
ON A Repository
Prenatal Diagnosis
Cell Culturing
Cytogenetic Analysis
-
-
27
T-3.bJ€' III.
Int€'grated Genetics: DNA Tests and Costs
DNA TESTS
PUBLIC CHARGE
CYSTIC FIBROSIS
- AF 508 Mutation Analysis
f'1e thod
Method 2 - Linkage Analysis
Method 3 - Disequil ibrium Study
-$225 per samp I€'
-$330 per sample
-$220 per samp Ie
DUCHENNE r1USCULAR DYSTROPW(
Primar)l Study
Ext€'nded Study
-$980 per sample
$360-$1210 add'],
per sample
SICKLE CELL ANEr1 I A
-$580 per samp Ie
ADULT POLYCYSTIC KIDNEY DISEASE
$360 per sample
HUNTINGTON-'S DISEASE
Presymptomat i c
Non-Disclosing Prenatal
l495 per sample
-t440 per samp] e
PATERNITY TESTING
Le'-}el
Level
DNA BANKING
-
I
II
-$165 per sample
-$285 per samp Ie
$65 per- sample
28
-
BIBLIOGRAPHY
Defesche, J.e., M. de Visser, E. BaKKer, G. Bouwsma, J.J. M. de Vijlder,
and P.A. Bolhuis. 1989. DNA restriction fragment length
polymorphisms in differ·ential diagnosis of geneti·c disease:
application in neuromuscular diseases.
Human Genetics 82:55-58.
Gene Screen.
2600 Stemmons FI,oJY.
Suite 133. Dallas, TX
Information folder, pamphlet.
Integrated Genetics Laboratories,
Massachusetts 01701.
Stine, Gerald J.
-
-
1989.
Inc.
75207.
One Mountain Road, Framingham,
The Nel,oJ Human Genetics.
Dubuque: Bro~l)n.
.
29
Huntlngton"s Disease
A Case Stud::-David,
a twenty-four-::--ear-old graduate student in the College of
Sciences and Humanities here at Ball State, is married and the father of
h'lo small children.
He recentl::-- learned that his father is afflicted
with Huntington's Disease (HD).
deterioration of his
o~l/n
He is now witnessing the prolonged
father.
In his search for information about HD, David has gained the
follQl.l/ing Knowledge:
Huntington"s Disease is characterized by a
progressive degeneration of the central nervous s::--stem (Stine 92)
leading to a loss of motor control, personal i ty changes, depression, and
dementia (Roberts 624); other symptoms include grimacing, gesticulation,
ataxic movement, finger twitching, and speech disorders (JablonsKi 153);
1 iKe
David's father, victims of Huntington's
usuall~
exhibit no symptoms
of the disease until their forties and fifties (Jab',JnsKi 153);
the
disease is caused b::-- a single dominant gene, found to be located on
Chromosome 4 (Stine 235);
the homozygous condition (having hllo dominant
alleles for HD) is not compatable with I ife, therefore his father is
heterozygous; since his father is heterozygous, David oossesses a 50%
chance of also being a heterozygote for HD, meaning that he, too, may
later deteriorate in the same fashion as is his father (Curtis 391).
After learning all of th's information, David has se'.}er-al decisions
to
maKe.
Recent ad\)ancements in DNA technology have led to a genetic
test which may determine whether or not a person at risk carries the
dominant allele.
David learned that the test was developed in 1983 by
James F. Gusella of
~1assachusetts
General Hospital (Roberts 624).
also beginning to real ize what a dramatic decision he must maKe:
He is
ought
30
or ought not I be tested to determine whether or not I will develop the
symptoms of Huntington's Disease?
David decides to do some research on the test itself before making
a dec i s ion.
I n fur ther research he comes acr'oss a study that was done
at Johns HopKins University concerning the test available for
pre-symptomatic diagnosis of HD and those who are at risk of developing
the disease.
Only 60 of 350 at-risk persons (17%) chose to be tested.
This adds more to David/s dilemma:
ought I have the test?
information do I need before I decide?
persons come up with their deciSions?
will lead me to such a decision?
this'?
what other
how did these 350 at-risk
what values do I possess that
where does my spouse fit in to all of
what about my children, ought they be tested also since they nOt.oJ
possess a 25% chance of also having the HD gene?
David again decides to get more specific information about the test
itself.
His main question concerns the test's accuracy:
is this genetic test?
how accurate
David discovers that researchers detected a
particual segment of DNA
(1..oJith
a par·ticular sequence of nucleotides)
that is inherited along l,oJith the HD gene.
This segment of DNA is
r' e f er' red t a a sag e net i c mar ke r t hat i s t r' an sm itt e d I ike age n e from
generation to generation (Stine 235).
the HD gene.
The marker flags the presence of
If the genetic marKer is present, the gene for HD is most
1 iKely present also.
Dat)id can now be tested by anabzing his
both of his parents using RFLP technology.
tis~.ue
.3.nd tissue from
If one parent is Known to
carry the gene and one is not (as in David's case, where his father has
the HD gene and his mother does not), then the presence or absence of
the gene in David's tissue can be determined by comparing the respective
31
lengths of DNA fragments cut using particualr restriction enz)'mes
<Curtis 397).
paternal.
Part of the fragments INil1 be maternal in origin, part
If the fragment containing the HD gene matches that of his
father"'s abarent
fr"~gment,
David carries the HD gene and later in life
will develop the same symptoms his father is experiencing.
However, David ascertains that the test is not 100% accurate.
Due
to the rare occurrence of crossing over which does occur in the
pol ymorph ism four percerit of the time, the marKer" may segregate
independently of the HD gene.
96~~.
The accuracy of the test is therefore
These data add yet another compl ication to David's dilemma.
"I,.)
i th
ne~1)
i nforma t i on comes new respons i b iIi ty - how,
~I)hen,
why,
and where It will be used, as well as the right to choose whether it
INil1 be used" (Stine 236).
As Dal)id thinKs about all of this
information he ponders the following ethical questio::s.
DAVID'S PLACE AND THINK ABOUT WHAT YOU WOULD DO.
PUT YOURSELF IN
Answer these questions
for yourself.
1.
What r"easons could you I ist to support having the test done?
to support not having the test done?
Would you have the test done? Why/ why not?
2.
What kinds of things would maKe you change your mind about
having or not having the test?
i.e. you may choose to not have
the test done now, but could anything change your mind in the
future?
3.
Ought this testing be required for Known possible carriers?
What values do you possess that support required testing?
-
reject the notion of mandatory testing?
results have on others?
that
What impact might test
i.e. If testing ought not be required,
ought future offspring of Known possible carriers be able to
32
seeK compensation from their parents if the disease is then
passed on to them?
4.
l,.Jh i ch fam i 1y members ough t
tested?
I,.'Jhat
to par tic i pa te
if you choose to be
Ought important members be required to participate?
if a pertinent fami 1/ member refuses to cooperate and your
diagnosis cannot be completed?
What if such testing leads to
information that other fami ly members may not wish to Know?
5.
I,~ho
should have access to the results of the testing? i.e.
doctor's,
6.
insur'ance ccompanies, participants, relatit,oes?
HovJ might test accuracy affect :.. our decision?
i.e.
if more
accurate or less accurate?
7.
Ough t gene tic counse ling be r'equ ired, and for 1,lIh i ch members of
the fami 1 y?
-
8.
I..~hat
·are the legal
implications?
Could one family member sue
another for not reveal ing information releO)ant to their
reproductive health?
9.
"Will
individuals be able to protect their personal
reproojuctio.)e freedom in an era of almost unl imited genetic
knowledge about themselves and their children?" (Stine 443).
The
issues presented in this case study of genetic diagnosis apply
to most genetic diseases.
Because of these
issues, many feel
that
genetic diagnosis is headed into a morass of social, legal, and ethical
problems that medicine and society are not prepared to handle.
a way to prepare?
Will we or can we, as a society, handle these
Is there
issues?
"The questions far outweigh the answers, and it appears that those
-
questions I,lIill grow exponentialJ:y' as the (nelN genetics) opens greater
areas of knowledge" (Stine 443).
33
.-
BIBLIOGRAPHY
Curtis, Helena and N. Sue Barnes.
1989.
Biology.
New York: Worth.
Jablonski, Stanley.
1969.
Illustrated Dictionary of Eponymic Syndromes
and Diseases and their Synonyms.
Philadelphia: W.B. Saunders.
Robert~.,
Lesl ie.
9 Feb. 1990.
Science 247:624-627.
Stine, Gerald.
-
-
1989.
Huntington.'s Gene: So Near, Yet So Far.
The NelIJ Human
Genetic~.•
Dubuque: Brown.
34
•
,.-:. !:::;
".';0_'
CLr~?ntly
there are -evera'
methcds e,ployed to detect
Rece~t
:i'"'
rec:o:i~i;:;ir",3r"[t
t
L
of
i3
n~w
r~str~cticn
te=hnc'oS/, DNA
2bncr~a'
?F~P
techn;1 og',' he),':=; 1 ed to a new (!iode iJf
[i:-,J(c
det?c:icn: RFLP,
andvancement
f-aS~2nt
pr~bes
lenst~
polymorphism.
In
are used to detect the presence
genes.
technol
~g~
tE·chnc:'c::::jJ.es in ": ",C<,t
di~fers
~riJm
other
g~ne
,t c:a,nr:Jt be appl ie
diagnose an individua' •
~he
dcotor or
to
screening
E\
POPLli at ion.
_ ~earcher must know
OU',;',t
PGlymiJr~hisms
can
J
e
.j I.e' t \'2 ',::
ted b- ·! '
restriction enzyme is used t
r
CLt
a
~~man
=hr~mcsome
at
2
"36
The resulting
particu1ar DNA sequence.
restr~~tlo~
ar? called
f~agment
E~c~
p:ec2s
~~stricticn
enzyme cuts
t~e
sequence; therefore, each r2striction enzyme
mut~tion
ex~sts
sequence
ThQ
~he
t~
at 3
be a1
t2r~d.
'0
t~e
0
t~
n :
some~i~es
If a
instead of
2re
1
c211e0
P~LP
pDlymor~hism
indep2~jently)
inked on the same
p~lymc
allcl~s.
stretc~
a
1 cut
~auuses
the DNA
~f
inherited diseases
'his~s,
or
DNA,
mar~er
These F'FLF' "Ci.11.:::>1es" de:; not
cosegregates
~i~~
W~l
a~
fragment length polymorphisms
a number
0'
Recogniz3ble DNA
are
O~A
.. s the seQuence is not recognized
res~riction
dete~tion
2NA
cuttIng site, the enzyme will
A mutation
::;ite.
presence of
(PFLPs) aId
p3rtlc_'~~
TJNA
~f
length colymorphisms, or RFLPs
specif~c
not cut
-
fras~ent
Csesrztes to;ether
a defective gers, then the two
of rNA.
gene.
even though the eXdct 'ocaticn of the defF:tiY2
ge~o
may
known.
able to
appe2TS.
de~ermine
For
C~
which chrom0somes a defective
exa~~le,
t~e
~untington/~
g2~e
gene aprear0 on
~ot
be
--
c~romo~~me
th~s
n2rr8wing
~~e
2~fort
to
~solatE
the g?ne itsel
L
how its m.::;.1 funct i on
By mid-1987 more than one thousand
This
r,;i::1S
,3
'~r-·ea.t
CO··~·fT:e~-·C1Er.:
defective hum3n
t~e
human genome .
.,
~~::t
.-
ge~es
;
iiT;P'3ct
(S~in~
1
Currently m2re than fifty
235) .
This information is also
38
Table I.
Example DNA fragments wi th restriction si tes.
A.
A DNA fragment with hllo restriction sites.
8.
A DNA fragment l..!Jith onl/ one restriction site; a mutation has
occurred at the s~cond site.
C.
A DNA fragment with only one restriction site; a mutation has
occurred at the first site.
(
/
I
/0'.
\
\.
,
\
"
\.
\
D.
.~
:/
"
I
_ ........
A DNA fragment with no restriction sites; mutations have occurred at
both si hs.
,-
Table II. Gene Screen: RFLP Related Services
TESTING OFFERED
Genetic Series
Alpha 1- Antitrypsin Deficiency
Cystic Fibrosis
Duchenne Muscular Dystrophy
HLA B27
Sickle-Cell Anemia
Type I Diabetes Mellitus
Identity Series
Forensic Testing
Paternity Testing
TESTING UNDER DEVELOPMENT
(
Cardiac Risk Panel
Bone Marrow Graft Monitoring
Leukemia and Lymphoma Diagnostics
Neurofibromatosis
Prognostic Test for Breast Cancer
OTHER SERVICES AVAILABLE
DN A Repository
Prenatal Diagnosis
Cell Culturing
•
Cytogenetic Analysis
,-
40
Table I I I .
Gene Screen, price 1 ists
DUCHENNE/BECKER MLSSULAR
PRICE LIST
1
DYSTRSP~Y
E~FECTIVE
TESTING SERVICES
FE8RUPRY 1
J
19Eq
Carrier Status Determination
Including up to s x family members or any
other members ~eqLired Lor diagnosis
Any additioral famil~
carr~er status
membe~s
$1400.00
$250.00
requEstIng
$1400.00
Prenatal Diagnosis
Inc·uding farri1; analy3~s of up to 6
family members or 3ry other rrembers
reqLired for diagnosis
Prenatal Diag~o~is
On a family preyious~y diagnosed by GeneScreen
Su~sequent
DNA Repository!Pank
(cer sample~
Inc~udes extraction of DNA 2rd stor~~e for
5 years.
This ccst may je app'ied
n Lu11
toward testing
Cul turing
From
F~om
a~niotic fluid
(per specimen)
CVS tissue (per specimen)
Analysis ('nc1uji~g culturin] for
Fron amniotic fluid (per ~Gecimen'
From CVS tissue (per sppcImen\
Cv~oge~eti=
$350.00
$50.00
t200.00
$275.00
~NA
a~21y~isl
$475.0C
$57500
41
Table IV.
Gene Screen, price 1 ist
CyaTIC
~IBROSIS
TES~ING
SERVICES
1988
Prenatal Diagnosis *
Informativeness Test Only **
Subsequent Prenatal Samp 1 e
Any add~tional £2mily members
for d i ct'3r-t':.:"-:, i,5 i per ~,amp 1 e) ~*,~
Car r-' i
t150.00
$850.oe,
E·j~' St at s
Deter-ITi i 11ati.-;:,r, ,)J;,*,*,*
Any additional ~amily mem~ers
for diagnosis (per sa~ple) ***
j,
Haplotype Analysis for Risk
Ir·-:L,viduai
P r-' e r' d tal
r::1!'~?'1
$725.')0
$]25.00
(per
5 c.:\ 'TIP
( inc 1 '-\ d: -: g
L'ep os ~~ t CT/ / 8,,::,1'1 k
Modi~ication
1e
b 0 :, !-
':j
c,:w' e :: t
:t150 • ;)(1
for Prenatal
$175.00
$450.00
5 )
$50.00
5 t:"JT'P 1 e)
(;F':I'-'
Diagnosis
Includes extraction DC DNA and
storage f~r 5 y?ars.
Tt:1S cost ,T,a,/ be ae'p'l ied in f,_,;,' 1
tCI--'~ard '~es+- in·::,:j "
eu: t'_lrinq
amniotic flUid
Fron CVS tissue (per
~rom
(~2r
$200.00
:t275.00
specimen)
specim~n)
Cytogenetic Analysis (including culturing for DNA analysis)
$4-7 5.00
~r~n am0iotic fluid
(per specimen)
$57'5,00
Fro~ CVS tissue
(per specime,l
, - - - , ----,-,--,,---------
*
Both parents affected child, and prsratal s~ecimen.
Any other fa.,T'L; y mi:?m~Jer's f:~l t neces~:'-;ji'-y to :Lmp;'ove
diagnostic accLwc"lC:y are '11 <::-0 inc1 uded.
*'**
',_.ar"ge C:~~ e:,:<::ende,:: fa;ni 1 ie:; ma-f be quoted
case D,::>,':; is.
****
Both parents,
~lus
CYI
a
af;2cted, p'us sibl ing for
~3peci':'il
diagn~sis
42
B I BL IOGRf71PHY
DeFesche. J.C., M. de Vlsser, E. Bak~er, G. Bouwsma , J.J.M. de
Vijlder, and ?A. So·huis.
1989.
DNA restriction fragme~t
length palymorphis~s in di'ferentlal diagnosis of genetic
diseas.e: appl icaticn i.n :-le'._tromusc'_.llar d::s.;eases.
Human
8:2 : 5':5--"":::3 •
Gene Screen.
75:207.
St ine
-
I
:2600 Stemmon= ~~y.
Information folder,
C3er'al d.
1989"
Suite 133, Dallas, Texas
pam~hlet.
Dubuque: Brown.
.-
-1-3
The di3ease is character:zed by a
progressive
det2r~~raticn
'.) i:: t ::. :T:S
dise~se
single
~ntil
C).[
!-iL'.n t .:. r-:'~'~ :)'-',
age
a~ter
dc~inant
al'e1~
t~2
of
=entra 1
'...(':: I. .!..~. ~ : '.'
I:;
e~.;
12cs~ej
to his
-
Cl~
he~
Dlsease\
2
Disease is caused by a
a parent who has HD
50~
c~ance
of passing
it on
offsprin
By the time the
£;~st
s~mptoms
?ppea~,
)~
,
:.he time cf h •
confirmatory
(Sti~e
RFLP technology to
~sing
~o=~e=ses
sy~tem
hi bit no symp t Clms:· of the
~~~tinJton/s
~0,
T~ere~ore,
~Huntington/3
nervous
the victim has often
charce of having
.
felt t,at
j.
f
infor~atlC)n.
It wasn't
until
ten
v~ars
~fter
t~e
dea~h
of
~is
Th is test,
-
developed by
Jam~s
two famil ies in
the
recog~ition
~.
~hi=h
of a
Cuse1'
3
Clf
~~ssachusetts
hurt::'ngtcr's
~a~ticular
Disea~e
segment
~f
General
existet
DNA that
~ospitd1,
result~d
is
in
i~her~ted
t~ansritted
genetic marker and is
gRneration
1 i~e a
be
~ow
~ested
for
ge~e
H~
RFLF'
i~
re~e~ber
that an31ysis
of no use in predicting
Gembers must
of a
g2~e
gene
ca~
a~so
using
be
o~
~rom
~s
g2ne~ation
a
to
is present, nost
by analyzing tissue
from the viet.:.:'" and bott-· parents using FWi._F'
important to
is referred to
~arker
If the genetic
(Stine 235l
Individuals can
DN~
This segment of
a'on9 with the disease.
techflc~
ogy.
It i'3
a single individual
using
Oth(-:?r f am i 1 Y
inher~tance.
te involved in order to determine the presence
~FLP
If one
a~alvsis.
by
deter~ined
cc~paring
DNA 'fragments cut U.SlnCj pa r ticl..ll
ar~
~0rent
is known to carry
the?spective lengths of
·?r;·~y;"ne~:
(Cu.-·t: s 397) .
Due tc]er".e
'nkage,
segment
if
~f
a parent passes on
DNA on which
that the HD gene wi 1 1
accuracy of the test
.£\150
t~e
gp,etic marker
gene is fourd'
be : :-1 h el'""' i ted.
is ne3rly 96%
After receiving "a11
with
~he
t~e
of this
..
:
1-::.. ).
(S~ine
(specific
~
r"o
i'-'en t 1 Y·;J
t
hI::?
2~6)
infarm2t~or,
Mr. Smith is faced
decision of whether or not to hale the test.
ago he would have
~ad
the test, but new
~e
has his dGubts.
His
f a.m i 1 y.
Results from a study at Jchns
~opkirs
Un:versity
that one year after notification, cnly 60 of 350
sh~we~
at-~is!
~erson;
k
45
(17~)
to je
c~c~e
Smith's
positi0~?
~
do
3UC~
~n
similar
d
/c~
If
1. "
do if you were in Mr.
~
PG~entjal
would you
wa~t
your parent to
case?
he usee, as well
-; t
·f
IOU
what would you want your spouse to
W~~t
situ~t.on
why, ane where it wll
--
w~~'d
were married to
vi~tim.
Huntington!s Diseas2
do in
What
~ested.
--:'tr:. ,_
~
,,:_.-lW,
C!ugh'c.
i:I-::S
r9l~tives
Wou'd you ask
Cug~t
fami~y
mem~er3
family nember =hcoses to be
~~
as the right to choose
lJ.Jherl mak i ng your
•
test.:Lr:C) be
~o
required to participate if a
test2~?
Who should pay Fer tpstlns of the
6.
Wr-,O sir OU! .::!
doc~ors
~e1
.'.:'. c: :~: 2' <::•. =:,. t '.:) the re '.. ."
r',3, \/e
c~
for known
rele.3e pertinent medical
5.
companies? The
re~uired
l .. , ..
tive~~
?
e'
The
relatives themselves?
...,
(
-
"
W~at
if ens of
the
-el~tives
test0 positive?
they be tDI cE'
8.
!O.
How might tl-Q prognosis affect
t~e
victim?
Would you w2rt to know th2t a rplative tested
,-" 1.,1--+·
;:; ......
~.-,
..
46
11
How would the know' edge
s9~t?nCe,
affect your
merr:b er-'s,'
Th:0ir bel",?v i.or-·r:·
Wh2~
abou~
~f
hav~ng
behavior~
~rena~2'
diagnosis:
,:,n::;.~ '/S~S
!:::e _.:sed to ".::'d if' tr,e.
of
t~e
Sh~uld
fs·tal
Wou1d the accuracy 0+ the test have an
14.
~ow
of
0:;:-
J. ...•
~~,~~
Who wil 1
3~fs=t
c~~n531
What are the legal
reproduct~ve.
freedc-n
., r
The
~,
genetic diseases.
E~e'=
(96X)?
behavio~?
i t be recommended?
impl i=a.tic!
In a time
if the
following the results?
Ought
l6.
What
i~pact
your jecisions and
~e~ple
tests
~arental
gf?r1c:-ne~
13.
wou'j
deat~
HG, a
pprio~
Cou1d one family
OL almost unl
~ffiite~
,
o.,
2Llse c)f
these
--
ethlc,31
''._U
h::?'id: e .
--
society, handle thesE iSiues?
that
t~ose
question~
~il ~
grow
2xronerti~11y
as the new
lenetlcs
47
:3 I 8L I CC3r::{'PH Y
Curtis, Hgler0
~.;orti-, .
a~~
N. SL2 Bar'2s.
Roberts, Lesl ie.
9 ceo
So r:- :3.1"-' •
~ i= _:. (7' It C ~;-.::.~
:989.
Gene:
r.
Dubuque: Brown.
-
-
48
~DDIT!ONAL
QUEST!ONS FOR DISCUSSION
GROUP LEADERS
FOR RFLP
1.
Would you have the t~sl done? Why or why not? What values do you
possess that lead to your decision? Do you have conflictin9 l}alue:·?
i.e. do some of your values support having the test done, ~lIhlle
other.values you possess do not?
2.
What Situations, technologies, etc. might lead you to change your
mind in the future?
3.
Ought testing be required? Why, why not? What are some of the
ramifications of mandatory testing? i.e. discrimination, quest for
the "perfect" person, etc.
4.
Ought you.a:.K relatives to release pertinent medical information to
aid in your RFLP diagnosis? Why might some relatives refuse? Ought
participation by pertinent relatives be required?
5.
Who should pay for the testing of the at-risK person and of
relatives involved?
6.
Who should have access to the results?
misuses of such information.
7.
How might the knowledge of having HD affect your behavior before the
appearance of symptoms? i.e. in David's case, hO~1I might such
information affect his behavior, activities, liest;/le nO~<J, before
the onset of degeneration?
8.
How would slJch information affect other fami 1;" members? If your
spouse 1,<Jere in Dav i d/ s s i tua t i on, how mi gh t your behav i or change?
How might your relitionship with your spou:.e change?
9.
Ought your two small children be tested? What about informed
consent, ought they be allowed to make that deci:ion on their own
when they are older?
Why? Name some possible
10. Ought prenatal tests be us~d to aid in the analysis of the fetal
genome at risK of HD? What if the fetus tests positive? Ought the
fetus be aborted based on such info? If not aborted, when ought the
parents tell a child that he/she will develop HD?
-50
~tudent5
Seventeen Sio 199
~3rtici~at~d
in
~he
disc~ssio~.
_-_
Yes:. ..'"7 il- .. 1..i~-
2.
Was t~e RFLP
comp'. .-·et"l'.=:r-::j . ..,
Yes 94. 1 ~/~
suffi~iently
~2terial
r~c!
for you
~ .~7~f.
Was enough information givEn or too much?
..... '-1,
1~ .. /~
W~re
you
y c~s ~~ 4 _" 1 ~.~_
give~
e~ough
r~c:~"5 "
In~ormation
3.
Dc ~o~ under3tand RFLP tec h n010gy and its appl ication after
readlng the m~te~:31 and at+2nCi~g the group discus~ion?
4.
Was the case stut~
y p.:; 94. 1 ~
!\Ie S. q~~
5.
Were you able
stud'l·:'
Yes
6.
2:':
II
1 ~~
\10
~o
.1.i·~1C·~,~
No
~~~~restirg?
~~entiry
2.
1 ()t)~.~
t~o
e~hica~
problem in the case
questions posed appropriate?
Q~/~
Was jour disc~s5ion group
qu?stions about RFLP~
f -"::::s:.
an
~~7..!~_
Are the ethical
Ye~.
-
in the case study?
91.
:~ader
a~le
to
an~~er
yOL~
()~/:
Please 1 ist th~ee good qual it~2S of the discussion.
-different ideas ~nd op~nions presented
-forced to think 0bout what I N0uld do
-interesting
-e~ua~
participation
-goo.j 1 ea.der
-looked at both sides of the issue
-may h?lp me ~ake jeci~ions iG the future
-made you think and put yG~rself in the situation
-learned about sLbJect
-exposed to other ways of t~i~kilg ~~out it
-di~ferent viewpoints
-pej·-·sor;a·1 i ;Ive ]'.'ement.
-more personal than class
-learned ~ore than i~ lect~re
-better understanding of materIal
-fe2' in3S about deat~ explored
--rc·e r-1a'/lng
-!,'.Ie- 1 p:·~epared
51
q
plea~e
~j
1 ist three
1. S C ,-l S SiD;:
suggestio~s
for
imprcvi~g
this
RF~P
•
-issues more pertine~t to stuje~ts' ~ ives
-relate RFL~ to more than just HU1tlngtcn's Disease
-more concentr2tion on pater0i~y testing
-suggest posstb'e ways to cure j~sease
_.,-j i. f f er-·ent. et hie ':';.1
::UE":ot i 0;-',·::;
-more role-p'ay~ng
-i~clude di~eases that can ~2 te~te~ for and treated
-·di;c .. l.·;:::,S dif'::erer;t ':-·::,s+-.s
-don't !nake i t=.;c; pr-2chctab: e
-r:on't make it so tec:i~0u.3
-''-;,Ol-'e sc eli ,:,\r i os
-spe~d less ~ime on ethical
~Jestions
10.
-
~td
your
Ye',"
16. :'::::;'
discussio~
No 23. ~l
go wel"?
Wh2~
pric~
kno~;edge
-'-k now 1 :;,~d 9
E'
t !-:,..- O~). s!",
yo~
jid
a~al;sis
-had used PFLP
i~
havo about
resea~ch
RFLP~
9xpe~ience
c 1 .:;', s·~,c.i2S
-in class
~nd
2.
Was the-e
enou~~
material
about RFLP provided to
3.
Did you -sad the
~?ter'al
on
res~~rch
e~perienc2
~eserve
you~
in the Science Health
;::;c ~t er- I': L~ L i h. '~ar
Yes /:J ~_.• 6 ~~
r-.~ C: ].~' ~1.~~
tI
If
-
4.
was i t
30,
Yes
~?lpf~l?
:!J~/%
Dij you
to
~,
,7' I,? t;-I
. __ ._
nee~
6.
?ny add_tional
-more articles on Rr~p
-var~2ti2n
in questi2ns
-Ne/a novie on RF~P
::::!~.::;
y·.-::')r
~~ e s
8.
research before the
!O~. sugge0t for preparing
for this discussion?
and cost
feel eomf,;rtab; e C:i.nd cC'nf ident beforE'
discuss :i. or\"
\-elL;
i :) ()~);
i'~c!
begi.n!iin·~
(} ~.~
Were you ab1e t~ identify
Yes 1001. No 21
wit~
the case
a~::l
st~dy?
e to to
:v!aybe ]?-=:3l ND O~
t:--,f? :::t _J:-2nl:~:; did ~;ot seem tc :jea]
emstions of the caSE.
Yes. {:l6..:...f:../-
COT"r:E"--lt:
-
9.
i 0 •
Were the
Y r?-:; t ()~)~!
eth~cal
Q'.-'er'd. 1 ·1
!-:'!\I'J
l\~ D
j
y
well;
,j .;.
d
on
app~opriate~
IC"'!'~~
- '.JS·3 i or gc;?
discussion,::
oj i~"
·::,t~e--·
"aei of attentic::n
-problems getting
. ·:\'~r-·E':2(j
~osed
t'V2
... ~~
--nct-':!s 1 i:(:,l y as·
-fait~l
questions
(.'Ji.th
t~e
1::he p"-'cdJl,::oms
students to t31k
f~eely;
they all
11.
W~at
factors do you think he'p0c your d~scussion~
cassed ou~ =n RFLP and HD
-gocj explanation of RFLP in paper
--the ~ackground infor~ation t~e ~eajer already knew
-i~for~2~io~
What Lactors hiGder~d your discussion?
-the age of the students, they don't 1 ike to thin~ abeut
d ,:?at I··.
-asked questiors t~at t~e leade~ could not answer
-5 tudent '::: ,j i d not-=:.ee hC~'J F:FL!::'""pp 1 i E'C t:-' thE'm
1:::.
~'Jhat
Su.·~ge5.t.:L:ms
~·m~_.l cj
"/'::'U :T:i:.i!-::e
-better ~eading 2r~icles
-suggestions for 'eaders fer
q~.est.ions
Wha~
to use in discussion
13.
suggestions would yeu ~a~e to improve the discussion?
-more pre~aration fo~ the leaders at t~e Bio 390 meeting;
how to deal wit~ the emotion2~ and mental ramifications of
F::F LF' c a O::i~' ~::. t u c:./
-~ake it app'y mere to stutents
-different, VarIOUS questions for ~he leaders to ask
14.
W~~
-
Y':?s
.-
to i.mpl-·ove the readi.ng?
the d:scussio~
~ 00:';_
No ;:I
wGrthw~ile7
Honors 2'79 RFLP Djs'::ussior, E·v.::;t.l
U,7:\t:L :W,3
j
Forti-five honors 299 stujent~ pa~ticipated
1.
Jvei~·.3.11,
97. 8~1.
Yes
w,,,,s· tl"~'~" I,\"-itt'?n
No =:. :-21.
Was the RFLP material
Yeo:; =3., 31.
~,Jo 6 "_T{
Did you comprehend the
Yes 5:'7 ::..:.8\
No 2. 2.l
T:iOl.ter"i..s.1
U?9~
Fal'
(".1
the disc~3s1on
in
eC!.r a.nd
unc'r'?rst,?nd3tl,-::,:,"':'
sufficIently explained?
m3te~ial?
Yes 7 ~ " 1 ~~
CQ,;1ment <;" ~
-give more sp2c"fic ~~formati~~
"'cou'd ;":.::\ve '::;~\,i2i" T..:J~-":? ethj.c:~~.l in·LJrm.3.tion
....··n,? Jt.:J e mDre i ,., f r:lt- :I~:'~ t: i err is n f:'c;,C ed
-
:3.
y:-:JUt~
Rat.;:?
Ltr":dE:T<::··' E'.rd i n9 :::+
clppl ica+-.ion a.fter·
, .-.
~-
,":-;:,:\dJ.;1g
F:F~.. F' technc' 0(3 y and it. s
the :-nate;''': :<.1 and at.tending
the
_., .•• -- -. -- .. - ..••..•.....- - .'" _.- .- ..- -- -- _ ••." •... --,- 'r
.1.
~
Mcej er
.:!..j .. :::?
gO(J~
1\,...1
e r'" ";/ G C' f.J d
:
M~~,d?r"ate
4.
5 •
t~e
Was
Yes
,,::?~~
.. 4 ~~
:>~
case study
'2.2,91.
VtJ e t' :;::
"'73
"/' c· L.t
\k
a t::r'!:?
interestirg?
9.1··~
t
:~:~
idE :'-, t :. ..}: '>."
~i n
~ t. ~-,
i, C .3 1
~l
j-- ;:)
b
~ ~'~T
::.
n
:~: h
e
C -:7\ ':-~. e
3t~td":'i''''
Yes 95.61.
6.
7 ..
No 4,41.
Are the ethica~ ~u9stior~
Yes 97.81.
No 2.~%
!.Ala's
/'C'L.i-
qr"Oltp
·:e.3.c~~r
at~:.~-?
posed
to
appro~~i2te~
·3.nS~"'Jer-·
···l::·!-.t~""
r::;:.~rc::t:.;~J""'·:::.
RFLP?
8.
OVE?~'all,
I lr40ul,j :"C:'l i:e this:. di::.cus-==.ici
Strongly agree
31.21.
A,-.:jf~:.?e
~··r
l' . ,
OJ. •
-'·t· ...11
Neutr~al
-.
r')1I,1
,;,
II
_.; ....
I'i. 5
Ll
a
5~1.
=''':3 ree
\"i~:'r-th"lhi;e?
3CfJLlt
1991.
1.
Wha~
p-ior
-Bi:J
; '?'::t
kn~wledge
did
yo~
have about
RCLP~
h er c 1 a:~7·(::!:;
-']enetics
'-0 +~
-I~'E:
,:;"d i r, ';
from Sam R~~ne
'ot of prior knowledge
-lec~ure
-2
2.
~ec~nical
-add more
:]
-,
irformat~~n
Was enc_gh
about
~nformatior
~a+er:al
Dld you read the
.; L.
-;
~FLP
provided to
abo~t
you~
how the procedure
on reserVE in the SHSL?
"'I
.
~....,~ .::.:..
V.J2.·::;
it
'(2S
J 00:'-::
h;:?~
i-)fui:i'~oY~
Was the written
mater~al
C122~
~nd
Rate your understanding of RFLP
ap~~
ication after
<) ",·r-'/:.:J ood
reading the
~nder~tandable~
tec~n:logy
3nd its
material
~rovidej
33. :J~~
'v;odE~r',,::<.te
6f:.1. 7f.
Very poor
Of.
7.
What other methods ~ou1j
OL suggest fo' ~re)aring
discussion group leaders for this di~=us~ion?
- 2 model
discl.:<::,sio:-: ']r-"::;'U;:' for' ::>ad""rs
--g i', e i r' '!: o!~ma t i.lJi~ ab DU, t t i": '::? u:-: ,~:; uen e ",5::' :'::In i. ,,,j i \1 i d ;';,::11 / S
DNA sequ0~ce 3nd ~O~ v0riations are not too much to c~ange
conserve~ sequences
o
-t.alk
-
ajoLtt
-RFLP
~eaders,
[!,~,:!
lab
Ye~:
bestje=:,
C::sea'7.:~e,
83. 3~·~
i
1 i~:e
Cr-'; T,':=!
too
::c'rr:fc·~-ta"!:Jle
and c:onfj,d=:::::nt befclre oegir:ni,")g
c: i 5,= L~ 55 i c)n '"':'l
Wer9
Yes
for
icc{,tl~::!i
materi~l
''jOLt feel
y'::JU,lr.
9.
appl
te~t~-g
-lecture
8u
C;t.i.'E'r'
and paternity
'\jCJ
JOG
able
(H)%
No
16.7%
-,,---,
t~
()~{.
identify with the
C~SE
study?
l'Jere the ,ileIT,b',-?rs
case'")
10.
11.
the ethi=al
We~2
f\~c
~
What factors
~:::J
YE?""
'.J~:
i
OO:~
">/GUi~
';Jr',:::Jup abl e
ques~i0ns
~.Js2d
"::.D
.Jer~",onc<.i
i:;::e t.he
3ppr=priate~
th:nk helpsc your discussion?
were helpf~l
a~ong st~dents made ~or good discussion
3e~~chi~g n0=essary above anj beyond the
y~~
-~dditi~n211uestions
-di5ag~eement
-dEeper
ab ··::!!.-·t i
-the
val~2
'~:!n
1 S~:;l.: 2
in
inf~rmation
t~e
12.
What sug~est~o~s would you ~2ke to improve the discussion
::::,.-. ttle ··~e,::'i.d i. ngs~'
--fT.:?!: '? mor~e r'ea' ~ 1 i. -+? -1 i. k ,? ~ r' :;(~C! -:0 get. same feel i ngs as
the per3cn ~n the case ~~ud!
-make the re0dngs ~~ t~e et~ic~l and legal dilemmas
13.
Overall
Stron31
--
j
I feel
agree
tnat. t~o
66.71
l\grE?E?
Neu1---'al
StrDngl~
-
arti=las on reserve in the 1 ibrary
disagree
0%
disc~ssio~
~as
worthwh~le.
-
58
DNA
RESTRICTION FRAGMENT LENGTH POLYNORPHISNS
(RFLP-S)
A READING LIST
Begly, Sharon. Leaving Holmes in the Dust. 1987. Newsweek. October 16. Page 81.
Cells that Convict. 1988. U.S. News« World Report. February 22, 1988. Page 11.
Couple Sues After Learning Dead Girl Wasn't Their Child. 1988. Associated Press.
Spokesl1J6/1 Review-Chronic/e.· October 9.
DNA Prints. 1987. Time. January 26. Page 66.
Fingerprinting DNA From a Single Hair. 1988. Science News,
February 3. Page 262.
Higuchi, R., ela/. 1988. DNA Typing From Single Hairs. Nature. 332:543-546.
Koshland, Daniel E. 1988. A Tale of Two Techniques. Science 242:993.
Lewin, Roger. 1986. DNA Fingerprints in Health and Disease. Science 233:421-522.
Lewis, Ricki. 1988. DNA Fingerprints: Witness for the Prosecution. Oiscover. June.
Lewis, Ricki. 1989. Genetics Meets Forensics. BiaScience 39:6-9.
Man, Jean. 1988. DNA Fingerprinting Takes the Witness Stand. SCJence240:1616-1618.
Moody, Mark. 1989. DNA Analysis in Forensic Science. BioScience 39:31-36.
Schmaltz, Jeffery. 1988. New York Times News Service. Spokesmll11 Revie1V-Chronic/e.
September 27.
Strauss, Stephen. 1988. DNA Fingerprinting. Techoology ReVIew 91 :8-1 0
ToufeIis, Anastasia. 1988. Convicted by their Genes. Time. Oct 31. Page 74.
Weiss, Rick. 1989. Doling out DNA. Science News, February 4: Pages 72-74.
Wilson, Mike. 1989. Associated Press. Spokesman Review-Chronic/e. May 7.
59
60
Lesson 5 What are RFLPs?
Part A : Direct Detection
Int.r .... ct.i ...
Lindo Fulcher and her hu~band Glenn lIere a bit neruou~ as they .et lIith
Beth Greendale, the genetic counselor at Children's Hospital.
The
Fulchers, a young black couple frail Mialli, had been l1Iarried for three
year~,
and noUl they wonted t.o ~tart a fall; Iy.
Like I1I03t young people, the Fulcher:s lIere healthy. They a~sull'led
that any children they might have Ulould be healthy, too.
But Ulhen they
heard at their· c:'urch about the screening progrol1l for sickle cell trait,
they thought the~ ;3hould iook into it.
They fOLlnd that about
in 625 black children in the United States
is born II i t h ' i ck Ieee I I anetJI i CJ olJch year. When Bet h Greenda Ie gaue her
present at ion t'J the church group as part 0 f t he screen i ng progra., sh~
said that sickle cell anellia is caused by a problell .ith he.oglobin, the
protein that carrie~ oxygen in the blood.
As a result of the abnor.al
he.oglobin, the blood cells take on a sickled shape, as shoUln in Figure
5-1.
The cells are long and rigid, instead of round and flexible.
People .ho ha~le the di~order experience severe pain in their long bones
and joints anel are susceptible to infections.
So.e do not live beyond
childhood.
Uith good lIedical care, hOlleuer, affected persons can live
reasonably nor.al lives into adulthood.
FIGURE 5-1.
Sickled cells.
61
Sickle ee! I' anemia, Beth ~aid,
i~ a rece~3iue trait. That mean~
affected indiuidual~ have tlllO abnormal gene~, one frolll each parent.
Som~ peopie, Beth "aid about 1 in 10 peopie in the biack population,
haue one gene.
They are ~aid to be carriers and to houe sickle cell
trait.
They heue na hee Ith probl em:!, but if they produce chi Idren .. i th
another carrier', there i~ a 2S percent chance that each child lUill haue
3ickle
cell
onellia.
Beth had
explained thot ec:ch :Dajor ethnic group eee!1l3 to heue one
that occurs more frequent Iy in that population.
tr'ace their Qnc~st('y to Rfei.:o, at'e at increo~ed ri3k for
di~order
geilet. i c
who
Diack~,
eel I
3ickle
an e ill i a .
\...lnda and r.;·,enn decided to ;"'Qve t.i"',~3eiue~ te3ted f'Jr 3·,cI. . le cell
Wt':en the r~~uit3 3ho!!.!~d t"ey ooth had '3ickie Gel; trait, they
coll~d Beth at the ge!'let!c coun3eling ciinic at Children's Ho~pital to
talk about family pll:1nning.
Beth told the!ll that there is a verq
accurate prenatal
te:Jt that can determine .. bethel'" the developing fetu:J
t.r-::P..
has sickle cell anemia, eick.le cell trait,
or norlllal helllc'giobin.
Linda and Glenn li,tened
a~
Beth
introduced
them
to BFLP:s.
,-
Red blo':ld cell~ are filled Ulith a ~otein called hemoglobin.
Hemoglobin car"ie, oxygen to cell~ that are actively \lork.ing.
In the
late 1940~, Oro. Linu~ Pauling ~ugge~ted that ,ickle cell ane!llia might
cau~ed by an abnormalily in hemoglobin.
R per~on with ~ickle cell
anemia ha~ a 1011 red blood count (anellia).
be
TABLE 5-1. The GenetiC Code
First
Base
I
A
or
U
G
or
i
i
C
I
I
I
AAA UUU}Ph
e
AAG uue
AAT UUA}
AAC UUG Ley
AGA
AGG
AGT
AGe
GAA
GAG
GAT
GAC
eVA
etiG
GGA
GGG
GGT
GGe
AUU}
AUe lie
AVA
AUG fMe~
TGA
TGG
TG7
TGC
T
or
TAA
TAG
A
T''''
"'.
e
or
G
II
TAe
CAA
CUUj
cue Leu
GiJU
CAG Gi..:C
CAT
CAC
Second Base
TorA
G or e
Aor U
I
GUA
GiJG'
Vai
eGA
CGG
eGT
eGC
UCUj
uee
UCA
UCG
Ser
1
ATA
ATG
ATT
ATC
UAU}
UAe Tyr
UAA}
UAG Stop
GTA
GTG
G77
GTC
CAU} .
C4e HIS
AAU}
AAG Asn
A~ I
AeG,
TTA
TTG
TIT
Fe
1
("",l.
GAUl
!.:>.so
G.4C
G.4A 1(j'
G.4G! 'IU
ccu
ece
Pro
eCA
eeG
1
Aeu
ACe }Tr.r
GCU
1"':. .......
I
""" Ii..:"".
GCA
GCG,
GC
Third
Base
Cor G
C4A}
C4G Gin
AAA\,
AAG J
-1'.
ACA
ACG
ACT
ACC
UGU
UGC}CYS
Go..
GCG
GCT
Gee
CGU
1
CGC
r9
eGG
TorA
Cor G
TCA
reG
TCT
Tee
AGUl
AGe ISe'
AorU
G or
AG.41
AGG fAr;
TorA
eor G
CCo!.
GG'-'l
G<:C I •
GGA (j,y
CCG
eel
eec
AorU
GorC
TorA
CorG
UGA }Sto p
UGG Trp
AorU
GorC
A
CGA
GGG)
e
I
I
Aor U
Gor
TorA
Cor G
e
Note: The DNA cedens acpear in regUlar type; the complementary RNA eocens are 'n' r
thymine. U _ uracil (reolaces thymine on RNA), In RNA adenine is eomoiementar t I t~ta ICS. A; aoenine. C a cytoSine. G - guanine. T of DNA; cytosin,e is cor.,piementary te guanine. and '.. i~e versa. 'Stop' a c:hai~ t yo ymlne.o DNA; ~raolls complementary to adenine
aoorevlated as TOllows:
ermlnatlon or nonsense cooon. The amono acids are
Ala
Asp - ascartic acid (jiu =
A - alanonl!
. .
Cysj- '
CYSteine
Gly
Asrg .. arginine' (
n .. asparagine
;r. a giutamlne
His ..
olutamlC
acid
-
= glycine
histldir,e
Ile~'Isole'uc·.·ne
Le'u .. leUCine
Lys = I"",'ne
7"
Met - methionine
p-'Ie .. phenylalanine
Pro .. proline
Ser .. serine
Thr = threonine
Trp .. typtopnan
Tyr ~ tyrosine
Val .. valine
-
62
Many yeClr~ of re~earch revealed that the DNA that code~ for the
helloglobin lIolecule ha~ a tlutation in individual~ .1'10 have ~j.ckle
helloglobin.
7he lIutation change5 one all1ino acid (giutalllic aCid) in
norllol helloglobin to a different ollino acid (voline) in ~ickle
helloglobin.
"ihat change give~ the red blood cell~ a different ~hape,
.hich cau~e~ the problelll~ ~een in affected individual~.
"aterial~
(per teall of tIIo)
paper cl ip~:
11
Procedure
1.
and
block,
27 .hite,
12 green, 22 red
Di~cu~~ion
The fo 110. i n9
helloglobin and
codon~
~ickle
are part of the DNA
hemoglobin:
~equence
for
norlla I
Codon Prate i n
GGT eTe CTC
GGT CAC CTC
Horlllal helloglobin DNA
Sickle helloglobin DNA
U~e the genetic code in Table 5-1
to deterlline the aaino acid~ that
are coded for by tho~e codon~.
De~cr ibe the di fference
in the
genetic code betlleen the ho types of hemoglobin. and the re3ult of
that difference.
2.
Strand~
A and 8 repre~ent ho ~ingle :!trond~ of DNA. U:!ing the paper
one teall lIellber lIill lIIake a .odel of ~trand A and the other
tea • •eaber _ ill .oke a lIode I of :!trand B on the next page.
Key:
black· adenine (A)j IIhite • thylline (T)j green· guanine (G)j red·
cyto~ine
(C).
clip~.
St rand
C;
2
A
A
3
"t
5
6
G
G T
C
7'
T
8 9 10 11
C C T
C
1a
A
R
T
T
G~
33
C
31 35
T T
15
T
16 17
T G
12
13
H
19 20 21 22 23 21 25 26 27 26
29
30
31
T
C
T
32
C
12
T
13
A
A
30
C
31
T
32
C
C
2
1
A
A
---- 19
T
C
C
T
3
G
"t
G
5
T
T
T
A
G
G
6
C
7
8
T C
11
9 10
C T
C
20 21 22 23 2., 2S 26 27 28
A C C T T A G G
C
Uhat
are
the
.ake-up?
5t rand
29
T
15
G
B
d i ff er-e nce:. in the ~t rand:.?
about on individual IIh 0
8?
.1'1 at can you predict
3.
16 17
T
T
St rand
~
A
Ii
8a~ed
ha:!
33
3., 35
C
T T
18
G~
on that di Herence,
A in her genetic
~trand
The re~tri,::tion enzylle nSTll1 recognize~ the DNA :!equence
GGTCTCC and cuts the DHA betllcen the f i r~t T and the f i 1"3t C of
i
cut
that
63
~equence, reading frail left to right.
What lIill be t.he length of the
frag .. ent~ (ho • •any ba~e:s) if :strand A i:s cut !lith MSTIII?
What .,ill be
the length of the fragllent:s (ho. lIany b~e3) i f ~trand B i3 cut IIi th
nSTIII?
How could you u~e MSTlII to di:stingui3h 3ickle hemoglobin from
normal helloglobin?
A3:sulle that Ms. Fulcher undergoe:s prenatal diagn03i3. The genet i c
coun:selor 3ho!l3 Ms. Fulcher and her hu:sband the re~ult~ of the OHA te3t.
(a) fragment.
In eaeh case, give the diagnosi~ for the developing fetu3:
~jze~ - 5, If,
10,6; (b) frag.ent ~ize~ - S, H, 10, 2f, 6; (c)
fragment 3ize3 • 5, 2~, 6.
_
f.
The title of this activity asb:
-Uh.t are HFLP:.?You have jU3t
u3ed RFLP~ to te3t for the pre:sence of 3i ck I e hemog I ob in.
RFLP
:stand3 for re3triction fragllent length polyllorphi~ul.
U:se your
k.nowledge of re~triction enzyme:s and variation3 in DHA 3eqlJence~ to
III" it e
a paragraph that exp I a i n3 the nOllle re3t.r i c lion fragment I engt h
poly.orphj~1I3.
(Hot-~o-helpful
hint:
polYl1lorphi311 lie an 3 "man!:!
forll~,·
in thi~ ca3e, II any different length~).
5.
What restriction enzYfle
linked
i nher i tance?
6.
HOIl IIOU I d heterogene i ty-d if ferent lIutat i on~ caU3 i ng
affect a diagno~i:s flade on the ba3i:s of RFLP3?
7.
Uhat
Building
•
•
ti3sue:s
Concepts
Hu.an DHA
of bases.
Ua I" i at i orl
for'
site~
•
can
be
pattern~
u:sed
Aeviell
for
the
might
thi:s
look
for
in
dOllinant
the
~alle
or
X-
d i :sorder-
diagnosi~?
type of
follolling
i:s highly variable;
you
coneept~:
no ho people have
in ba:se sequence:s can create
restr iet ion enzy.es.
01"
the :sane
eli.inate
~equence
recognition
If lie digest (chop up) the DHA fro. ho people lIith the :salle
enzy.e, the variation in the DNA ui 1\ result in
di fferent sized fragllent:s for each person.
re~triction
•
These
frag.ent~-ca
di~ea~e-cau:sing
•
II ed
AFLPs-can
be used
to detect
the
presence
of
gene~.
The genes for sane di :sorder~ ~uch a3 3ickle ce!! ·:memia can be
detected directly.
That requires knolJledge of the :5peeific
.utat i on and a restr i ct ion enzylle that cut:5 DNA at the Slut at i on
sit e.
Pert B Linkage
Intra4.ct i en
,-
Karen not iced .any neu face~ in her lOt h grade hOlleroom., but lIa3 3t ruck
by one boy in particular.
He lIa3 ~.aller than 1I0~t other bOy:5 in cla:5~
and ~ee!led very th in.
He had br ight. brown eye~ that ~eemed to not i ce
everything and he wa3 quick to laugh at anything funny.
R:s the ~chool
year progre3~ed, (aren learned hi~ na.e lIa3 Ti. and got to knOll hill
64
a~
pretty \lell,
they
had
Engli~h
and
Spani~h
cla~~e~
together.
One day Ti. wa~ ab~ent froll ~chool and Karen rellellbered that he had
been coughing a lot the preuiou~ tlUO da~.
Uhen Till did not return to
~choo I for a I ,.o~t t 1110 week.~, Karen got worr i ed.
She .a~ p I ea~ed .hen he
finally returned, although he looked a bit pale and ~till had a cough.
Karen caught up to hill after Engl i~h clas~ and ~aid "Hey, uhere'ue
you beenr
Oh,
had pneullonia for awhile," said Till . • , have cyst i c
fibro~i~, and I get pneullonia becau~e of it, ~o.etille~.·
Karen knew
about pneUilonia, but ~he had never heard of cy~tic fibrosi~.
·Uhat'~ cy~tic fibr03i~," ~he asked .
• We I I ," ~c i d T i til, • it's hal" d t 0 ex p I a in.
It's an i nhe r i t ed
di~order that ilcke~
it. herd fer me to digeet some foode.
It oleo
.y lung~ to clog up, ~o it'~ hard to breathe ~olletille~.·
I ~ that
nodd ed .
·You
-
why you've excused
inherited
it
from gyll samet i lIe~?·
fro II one of your parents then,"
~a
Karen.
Till
i d Karen.
·It'~ Q recessiue disorder,
~o
irflerited one cy~tic fibrosi~ gene
froll each of .y parent~.
They're carriers, but because it'~ a rece~siue
condition the!~ don't haue cy~tic fibrosis thell~elues.·
It lIIas tille for their next class, :xl Karen said goodbye to Till.
That night ~he began thinking about Till.
She lIentioned Till and his
problells with cy~t ic fibrosi~ to her lIother, who said she thought she
had seen a recent article in the local newspaper about cystic fibrosis.
She suggested Karen ask the I ibrarian about it.
The next day, Karen
found the article, which "as titled -C._tic Fi •••• i . S . . . . . . . . ..
t. Ch •••• s •• e '1.The article reported how a group of scientists,
led by Dr. Lap-Chee Tsui in Toronto, discouered that the cy~tic fibrosis
gene is linked to RFLPs on chrollosolle 7.
It went on to say that it
would nOli be po~sible to do prenatal testing for cyst ic fibrosis and
that this breakthrough lIight eventually lead to isolating the gene for
the disorder.
That sounded exciting to Koren, and she set out to learn
lIore about how I inkage is related to AFLPs.
Linkage and recollbination.
If you
acorns, you kllou that an oak tree
presence of acorns to the prfsence
object known to be associated with
-
a~ked
cause~
walk through a park and ~ee sOlie
is nearby.
You have I inked the
of one object b!:j identifying a second
the first.
Linked genes are found on the salle chrollosolle.
During lIeiosis,
chrollosolles cOile together in pair~.
The chrollosolle 7 that you inherited
froll your Ilother .ill line up next to the chrollosolle 7 that inherited
froll your father, and so on, . for each nUllbel"'ed pair.
At this tine,
paired chrollo:solles lIay exchange segaenls of DNA (Figure 5-2).
That is
called cros~ing Quer.
Thus, a segnent of OHA fl"'oll the chrollosolle that
was inher ited frOIl the lIa Ie parent lIay becolle attached to the chrollosoae
that was i nher i t ed fro" the feaa I e parent. The fert iii zed egg,
therefore, will contain lIaterial that has been rearranged by crossing
over.
This rearrangellent re~ult~ in di fferent sequence~ of genet ic
rlaterial, a situation called recollbination.
65
nutation3 and pal!la.rphi3a:s.
Biologi~t~ can detect
DNA ~equence~ on the ~aRle chromo~orae in different people.
I i ~hed b~ u~ i n9 the ~olle AFLP techn i que~ ~ou I earned about
in part A of thi5 activity.
In pert A you ~all the effect of a raul at ion,
.here a change in one nucleotide re~ulted in a change in the protein, in
thi~ ca~e, helloglobin.
It al~o affected the nuraber and ~ize of RFLP~,
and that inforllation helped to deterlline whether the gene for sickle
helloglobi n .a~ present.
difference~ in
That i ~ OCCO!!lP
There ere changes in DNA sequences thet have no effect on the
protein produc-:.
The~e are called polyllorphism~.
Consult the genetic
code in Table 5-i on page 34 to find the DNA codon3 for glutallic acid.
Explain hOIl a change in one of the base~ can sti II result in glutamic
acid.
HOll lIi~lht the sallie change affect the RFLP~ produced by a
restriction enzyae?
CC-iijiij'l
•
_~
(
'~
',/,")
C
Of-~::ij_:
,.
-
Si::;!: ;:air
chromosomes
-
Replication
FIGURE ~2.
,-
Crossing over during early stages of meiosis.
.at er i al3 (per tea. of tuo)
paper
clip~:
10 black,
23 white, 9 green,
16 red
Each partner .ill build one of the tuo follo.ing strands of OHA.
These are the ~alle segllents of honologou~ chro.oso.es fro. the sane
person.
1.
Strand
A (fran
chro.o~olle
lIother's
7)
1 2 3 ..
5 6 7 8
9 10
11
12 13 I i
15
~G___
G__T___C__T___T__C
___C__G
___T___C
____T___C___T
_____
C~
(T16 17161920212223 2i 25
~
eTC
eGG
B ( fran
St rand
2
r
-
G
G
3
T
~
5
C
A
6
T
"
A T R
7
C
The
restr iet jon
GGTCTTCC and
l' cut
27
28
29
30
T
R
T
R
T
father's
6 9 10 11
C
C G T
16 17 16 19 20 21 22 23 2i 25
'-;-1 T C T C C A G R T R
2.
26
chronosolle
12
T
13
C
7)
1't
IS
T
C -"
29
30
T
~
26
T
27
R
28
T
R
enZyille Matll I recognjze~ the DNA ~equence
bet.een the f i r~t C and the ~econd T.
cut~
-
66
3.
re~triction
The
enzyme
n~pl
recognize3
the
and cut~ beheen the ~econd C and the f ir3t
DNA
~equence
CCGG
tcut
G.
t.
Exalline your .odel~ of DNA ~trand~ A and B.
IJhat ~ize frag"ent~ (ho.
IIany ba~e~) .i II re~ult i f ~trand A i~ cut lIith M~t III and M~pl?
Uhat ~ize fl"ag.ents (ho• • any ba~e~,) .i II re~ult i f ~trand B i~ cut
.ith M~tlll and M~pl?
5.
Earl ier you lUere told that the~e DHA strand~ are segllent~ of
hOllIo loghou3 chrollo:5ome3 froUl the sat:le j:)er~on.
Uhet rea~on~ can you
find for the difference in fraglllent length3 betiJeen strands A and B?
A~su.e that
recognition site5 GGTCTTCC and CCGG are clo~ely linked.
Ho. could you u~e ju~t one restrict ion enzy.e to preduct the
pre5encc of both reeogn; t ion 5; te5?
6.
Earl ier
you
chrolloso.e~
learned that recollbination (cro~~ing over)
can take place during lIeio:!is. Revie. that
bet.een
proce~s
paired
if
necessary.
7.
A~~ulle
15
-
.ake
8.
that a break occur~ beheen b~e~ 15 and 16, IJ ith ba~e~ 1 to
over fro. ~trand B to strand A.
Use your DHA IIodel~ to
Hri~ crossover.
cro~sing
Uhat ~ize fragllents .i II re~ult if ~trand A i~ cut
nspl?
HOll does that co.pare to the fragllent froll
cross i n9 ouer?
uith
~trand
n~tlil
and
A before
9.
Uhat size fragllents .ill result if strand B Is cut uith "stili and
nspl?
HOIl does that co.pare to the fragllenh froll ~trand B before
cro~s i n9
over?
10.
In the ne,:t part of thi~ activity, you .ill inve~tigate ~olle
i.portant things lie can learn 'about the genetic~ of indiuidual~
us ing the AFLP techni que and the understandi n9 .e have of cros~i n9
over.
Detection
of
Cy~tic
Fibro~i~
You just dellonstrated that one recognition site IIay travel .ith a
different, but nearby, recognition ~ite becau~e of linkage.
In
addition, you learned that .olecular biologist~ can u~e the presence of
one site to pI-edict the pre~ence of another.
It tllO recognition sites
happen to be on the ~alle chro.oso.e. they lJi I I be i nher i ted together
unless there i~ reco!!bination (cro~~ing over) bet.een the. during
lIeio~i~.
-,
The frequency of recollbination bet.een tlJO linked recognition ~ite~
depends on the di~tance beheen those ~ite~.
If the sites are close
together on the chrol\o~o.e, reco.bination IJ; II be rare, and they .i II
alllo~t al.c!:l~ travel
together.'
If one ~ite happen~ to be a .. utation
site for a genetic disorder such cs cystic fibrosis (CF"
.e ccn follow
the i nher i tance of the CF gene by fo II ou i ng the i nher i tance of the
nearby recognition ~ite.
That .ill be true even if the ~econd ~ile ha~
67
-
,
nothing to do lIIith CF. Thi~ proce~~ tak.e~ advantage of
detectable poly .. orphi~ .. happen~ to travel lIith, or i~
particular gene.
The follolling procedure delllon~trate~
polyllorphi~1I i~ u~ed to detect the gene for a genetic
1.
the fact that
linked to, a
ho. a I inked
a
di~order.
The gene for CF is on the salle chroaosolle as a Hindlll recognition
and a n~pl recognition ~ite.
There i~ a recollbination bet.een
the Hindlll :site and the CF gene 15 percent of the tille.
Reco.binat ion bet.een the M:spl :sile and the CF gene occur:s only
percent 0 f the t i lie.
~ite
2.
The fo Iiolli ng I ine
the tao recognition
repre~ent~
~ite~
and
that part of a DNA ~trond occupi ed
the CF gene (the three dot~).
2
by
3
e ______e ______ e _________ _
If 1 i:s the
IJhat i ~ t he
3.
location of the CF gene, .hat
i ~ for your an~.er:s?
i:s
I
CF
gene
1_
other
other
bo :se~
CCGG
ba:se~
........_ _ _....J...
Uhat .ould
The follouing
a per:son "ho
Ti.'~
diagra.
not
doe~
illu:strote:s
have CF.
.hat
--CGG~ baoco
ot~er
other
a
diagrall of the chro.o:so .. e~
I
CF
I
--other
~
it e
I ike?
one chro.o:so.e
7 look:s
1
TAGCTT ]
like
other
baoco
------- -------
Make a ~i.ple diagrall to illu~trate the
expect to find in Till' ~ father.
i~
other
ba~e~
Hind I I I
----------~----
Here
'__
I
AAGCn
:s ite
other chrOIlO:SOlle 7 look
other
basco
-..
i:s :site 3?
----~--------~
n~p I
5.
27 IJhot
in the introduction to thi~ octivity you lIet Till, .,ho ha~ CF.
If.e
.ere ab Ie to look at a port ion of chrOIlO~Olle 7 fro. Ti II, .e lIoul d ~ee
the follolling:
other
ba:se:s
~.
~ite
ba~
froll
CCGG
I
chro.o~olle:s
Till'~
other
ba:se:5_~_~a_:s_e_:s____~___.__ ~~
M:sp I :5 i t e
you .ould
mother:
J
AA GCH
H j nd I I I
ot her
ba:se:s
~
it e
in
68
otherJ
bc~e~
normal
gene
other
CCRG
other
ba~e!S
bc~e~
----
TAGCTT
J
other
ba!Se!S
.~-.-
Where do the!Se ~equence!S di ffer fro. tho!Se on the father'!S
chro.o!Solle!S?
Uhich chro.o!Solle!S did Ti. inherit fro. his parents?
6.
Each 0 f Till.' s
chro.osolle '?
7.
Ue nOli knoUJ the recognition sites for chro.oso.e 7 in three
generotion:s of Till'~ fOl1ily.
Geneticists can use this infornation
predict the phenotype:s of other fanily lIIellber!S.
a.
grand fat her!S ha!S t he sequence CCGG -- RAGCTT on one
Uhat can you predict about Tia's grandfather~?
to
Till ha~ a ~i:ster, Ji II.
Before Ji II wa:s born, her nother unde":.ent
chorionic uillu:s biop~y.
An ob:stetl"'ician I"e.oued !So.e fetal cell~
fran the uteru~.
Genet ie i~t~ then studied the DIiA froll the cells.
They found the follolling:
-:::-rr.O:h~r
othel'"
ba !Se:s
CCGG
base!S
------
ba!Ses
-
rr.~h.r
ba!Ses
othel'"
ba!S e!S
-.~---------~----------~---
Chl"'o.osolle
othel'"
ba!Se!S
RAGCTT
1
CGGG
76
othel'"
base!S
TAGCTT
other
b a!S es
9.
Refer to the diagra.s of chl"'olloso.e 7 froll Jill's parent!S, in !Step!S ..
and 5.
/.Ihich parent contl"'ibuted Jill'!S 7B chl"'o.osone?
Uhich parent
contributed Jill's 7A chl"'olloso.e?
Is Jill's 7A chrollo:sone likely to
haue the CF gene or the nor.al gene?
Uhy?
Uhat can you predi ct
about Jill and CF?
10.
Once genet. i c i !Sts know the frequency of Cl"'ossouer bet ween d if ferent
region:s on a chro.oso.e, they can be specific in theil'" pl"'ediction!S.
At the beginning of this procedure you learned that the fl"'equency of
I"'eco.bination between a CF gene and an nspl I"'ecognition site is I
pel"'cent.
The recollbi nat ion freauency between a CF gene and a Hi ndlll
recognition oite i~ lS percent.
The~e
frequencie~ and carrier ~tatuo
can be used to pred ict Ji II's genotype.
What is the probab iii ty that
Ji II' s chro.o~ome 7A has the CF gene?
The 1'1 (nornal) gene?
Uhat is
the prabability that Jill's 76 chrolloso.e has the tf gene?
The CF
gen e?
.
11.
Because of recoabinat ion, Ji II has foul" po:ssib Ie genotypes:
7A CF
and 7B Hj 7A CF and 7B CF j 7A Hand 78 1'1; 7A 1'1 and 7B CF.
Use the
probabilitie~ you calculated in ,tep ;0 to deterllline the probability
that each of the four genotype~ li~ted will OCcur in Jill.
Overall,
what are Jill'~ chance, of being a carrier of CF, being norllal
or
J
ha'Jing CF?
In 'tep 9, you predicted Jill 1I0uid not have CF, but
be a
carrier.
Shc:uld lIodify your prediction ba~ed on the preceding data?
Uhy or why not?
'.-
Bu i I di ng
coneept~
Rev iell
the
fol lOlling concepts:
• linked.
Piece~ of DNA that are on the 3aae chrollo30lle are
•
•
•
. During lIIeio~i~, ho.ologou~
called crossing over.
chrolllosolle~
~ai d
exchange DNA
to
be
in a proee33
During croS3ing over, pieces of DNA that are cl03ely linked will
be
le~, frequently than piece, of DNA that are di~tantly
link~eparatej
ed.
SOlie
AFLP~
are cl03ely
I inked to
di~ea3e-causing
gene!'.
•
If one can detect ~uch an RFLP J
cau3ing gene 0130 is present.
•
The predictive power of linked AFLP~ depends on ho" cl03ely
they are to the disease-causing gene.
one can predict
that the diseaselinked
•
Linked aari(er3 allow detection of disease-causing genes in the
absence of knoll ledge about the underlying cause of the disea3e.
•
The ability to detect disease-causing genes prenatally and in
heterozygotes raises illportant ethical and legal quest ions.
Thi slab exerei se taken frOIl Advances in Genet ie Technology By
BSCS
and published by D.C.Heath who gave us perllission to use this
lab exercise.
69