2/12/16
BIOLOGY 207 - Dr.Locke
Lecture#28 - Restriction Fragment Length Polymorphisms
(RFLPs) -1
Required readings and problems:
Reading: Open Genetics, Chapter 8 & 10
Problems: Chapter 8 & 10
Optional
Griffiths (2008) 9th Ed. Readings: pp 14-17, 51-53, 146-153
Problems: 9th Ed. Ch. 20: 19, 21
Campbell (2008) 8th Ed. Readings: Concept 20.1, 20.2 & 20.4
Concepts:
What are RFLPs and how do they act like genetic marker loci?
1. Some mutations alter the distance between adjacent restriction enzyme sites.
Such alterations result in Restriction Fragment Length Polymorphisms (RFLP).
2. RFLPs represent allelic forms of a locus and behave as alleles in inheritance.
3. The location of each RFLP can be mapped genetically and thereby used to create
maps of chromosomes and genomes.
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 1
2/12/16
Restriction Fragments in Large Genomes
Over view:
Steps:
Genomic DNA:
Chromosome
---------------|---|------------O--/
\
/
\
/
\
Cosmid clone
/
E E
\
____|------|--|--------|___
/
\
/
\
/
\
plasmid subclone
/
\
]------------------[
The chromosome, cosmid clone and plasmid sub-clone have the same restriction
fragment.
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 2
2/12/16
Isolation of DNA from:
(1) genome, (2) cosmid clone, (3) plasmid clone
Each can be digested with a restriction enzyme that cleaves the double stranded DNA
at each site recognized by the enzyme.
This digested DNA can be separated on an agarose gel.
DNA
origin --> electrophoresis gel
___|_______________________
Plasmid clone
|
|
two sites.........|
0
| |
| vector + inserts
cosmid clone
|
|
many sites........|
0
|
| | | |
| vector + inserts
genomic DNA
|
|
many, many sites..|
0 |||||||||||||||||
|
genomic
|
|
size markers......|
0
| | | | | |
|
|___________________________|
Digest all with same enzyme (e.g. Eco RI)
Do Southern Transfer - DNA transferred to membrane.
Southern Transfer:
Probed with 32P labeled unique insert DNA - hybridized to:
1. sub cloned insert in plasmid + insert - not to vector
2. cloned insert in cosmid + insert - not to vector or other fragments
3. restriction fragment present on chromosome in genomic DNA - not to any other
fragments (unique DNA probe)
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 3
2/12/16
Autoradiogram of Southern blotted gel
size markers
origin
___|______________________
|
|
|
0
|
|
|
|
|
0
|
|
|
|
|
0
|
|
|
|
|
0
| | | | | |
|
|__________________________|
| 2 kb
plasmid
vector + inserts
cosmid
vector + inserts
genomic
Notes:
1. Hybridization intensity is proportional to the amount of insert DNA (to which the
probe can hybridize).
2. Thus, using a labeled probe and genomic DNA, one can identify the size of a
restriction fragment at a particular site in the genome.
3. Diploid organisms have homologous chromosomes, thus the genome has two
similar sequences (genes) at each locus -> 2 alleles. These two alleles may be
the same or different sized fragments.
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 4
2/12/16
Hypothetical Example:
2Kb
3Kb
Homolog
gene a+
E
E
E
1
---|--------|------------|--------|----------|----Homolog
gene a2
---|--------|------------|-------------------|----E
5Kb
E
alleles
|-probe--|
Autoradiograph of __________________________
Southern
|
|
Blot
|
0
|
|
|
|
|
Size markers
|
0
| | | | | |
|
|__________________________|
|
|
|
origin
5
2
kb
genomic DNA
size marker
In this example: loss of middle Eco site is due to an ancestral mutation that changed
the GAATTC sequence on homolog 1 to an unrecognized sequence (e.g.
TAATTC) on homolog 2 -> (no cleavage). Or visa versa.
The difference in the DNA sequence between the homologs makes the RFLP on each
homolog an allelic form.
Allele#1 = has the Eco site
Allele#2 = lacks the Eco site
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 5
2/12/16
RFLPs act like alleles in inheritance
On a genomic southern blot, the 2Kb Eco probe can detect an allelic difference.
homozygote
heterozygote
homozygote
Homolog
type
1/1
1/2
2/2
Fragment
size(s)
5 kb
5 kb*
Fragment
size(s)
2 kb *
2 kb
* = (double intensity because there are 2 copies per nucleus)
origin
___|________________________
|
|
|
0
|
|
|
|
|
0
|
|
|
|
|
|
0
|
|
|
|
|
0
| | | | | |
|
|___________________________|
|
|
size markers
5
2 kb
homozygote 1/1
heterozygote 1/2
homozygote 2/2
size markers
The "phenotype" of the allele is the size of the restriction fragment that hybridizes.
This is co-dominant because both alleles show up (no dominant or recessive).
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 6
2/12/16
The inheritance of these alleles is exactly like any other.
Just as a+ or a- would be passed on, so would the 2Kb or 5Kb restriction
fragment size as a phenotype (that directly reveals the genotype).
P1
2Kb/2Kb
F1 (P2)
F2
Ratio
2Kb/2Kb
1
x
|
2Kb/5Kb
|
2Kb/5Kb
2
5Kb/5Kb
5Kb/5Kb
1
2Kb and 5Kb alleles represent two different forms, or alleles at this "locus".
The "locus" is the middle Eco site, where the DNA sequence differs between the
homologous.
Polymorphic locus – multiple alleles of a locus exist in the population
For RFLPs, there are many different restriction fragment lengths
due to +/- sites or DNA insertions/deletions.
Remember a diploid individual can only have two allelic forms, one for each homolog
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 7
2/12/16
Degrees of Polymorphism
Some sequences are not polymorphic (monomorphic)
- - essentially everyone in the population has the same sized fragment.
- - (e.g. sequence encoding a protein - selection)
Other sites are highly polymorphic,
- many size fragments (alleles) in the population.
(e.g. regions between genes - little/no selection against mutations)
- often have repeats – variation in the number
------------------------------------------------------------------------------
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 8
2/12/16
RFLP can be mapped genetically
From previous example:
2Kb
3Kb
Homolog
gene a+
E
E
E
1
---|--------|--------|--------|----------|---Homolog
gene a2
---|--------|--------|-------------------|---E
5Kb
E
alleles
|-probe--|
Have two loci: (gene a) and the (Eco site RFLP) that each have allelic forms
Do standard genetic cross:
P1 a+ e+
a+ e+
F1 a+ e+
a- e-
X
a- ea- eX
a- ea- e-
test cross
Score progeny for a+ or a- (assume a+ is dominant to a-)
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 9
2/12/16
Test cross results:
Score all progeny for a+ or aAlso score for Eco site by Southern blot of each progeny-> test for 2Kb or 5Kb or both
Progeny:
Parentals
Independent Linked
a+ e+/a- e2/5
a- e-/a- e5/5
Recombinants
a+ e-/a- e5/5
a- e+/a- e2/5
If a gene and Eco site are close to each other then:
Recombinant Frequency will be low (RF=low)
-> few crossovers between a and Eco site.
If a gene and Eco site are distant from each other then:
Recombinant Frequency will be high (RF higher, approach 50%)
-> many crossovers between a and Eco.
By extension RFLP can be mapped relative to each other by using two different
probes and scoring appropriately for the respective phenotypes (fragment sizes).
 Advantages - next lecture
Biol207 Dr. Locke section
Lecture#28
Fall'11
page 10