DNA Technology
A.P. Biology
Biotech Intro
Glowing Fish
Glowing Mouse Mistake
What’s Next – Google Brain?
Uses of DNA Technology
Find out what genes do (mutate or knock
out a developmental gene and see what
happens)
 Make large amounts of a protein
 In-situ hybridization – can tell if an embryo
has a defective gene
 Diagnosis, treatment, prevention of
disease
 Study of relatedness of species
 Crime Solving
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DNA Technology Uses Continued
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Study gene expression
Study growth and differentiation
Identify recessive alleles
Vaccines (make large amounts of proteins that
trigger the immune response
Designer Drugs (anti-fat drugs)
RNAi (blocks translation – find out what a gene
does)
Gene Therapy (put genes in somatic or germ
cells)
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Problems:
How do you introduce it?
How do you control gene expression
How do you get the gene product where it’s needed
May alter other cell functions
Eugenics are a worry (controlling the genetic make-up)
Technology #1 – Gene Cloning
Gene Cloning
Making multiple copies of a gene by putting it in a
cell that will replicate it and pass it on to offspring
Although you can make lots of copies of a
gene in a tt, it will actually produce the
protein if in a cell
To clone a gene you will need:
restriction enzymes, a vector, and a host cell
Restriction Enzymes
a.
naturally used by bacteria to digest foreign
DNA
b.
Bacteria methylates its DNA (A&C) to
protect itself since methylation prevents
DNA digestion
c.
Can buy and use to cut and insert foreign
DNA
d.
Cut at palindromic sequences – same
forward and back (same 5’-3’ or vice versa)
Enzyme
Organism from which derived
(cut at *)
5' -->3'
Ava I
Anabaena variabilis
C* C/T C G A/G G
Bam HI
Bacillus amyloliquefaciens
G* G A T C C
Bgl II
Bacillus globigii
A* G A T C T
Eco RI
Escherichia coli RY 13
G* A A T T C
Eco RII
Escherichia coli R245
* C C A/T G G
Hae III
Haemophilus aegyptius
GG*CC
Hha I
Haemophilus haemolyticus
GCG*C
Hind III
Haemophilus inflenzae Rd
A* A G C T T
Hpa I
Haemophilus parainflenzae
G T T * AA C
Kpn I
Klebsiella pneumoniae
G GTAC * C
Mbo I
Moraxella bovis
*G A T C
Mbo I
Moraxella bovis
*G A T C
Pst I
Providencia stuartii
CTGCA*G
Sma I
Serratia marcescens
CCC*GGG
SstI
Streptomyces stanford
GAGCT*C
Sal I
Streptomyces albus G
G*TCGAC
Taq I
Thermophilus aquaticus
T*CGA
Cloning Vectors
Something to carry gene so it gets copied
a. Phages (as the phage replicates inside the bacteria so
does the added gene and it spreads to other bacteria)
– holds up to 25kb of DNA
b. Plasmids – as bacteria reproduce –
gene within the colony – holds up to 12 kb
clones
c. Retroviruses – have advantage that they can
incorporate into the host chromosome in animal cells –
Can hold 8-10kb
d.
Yeast artificial chromosomes (YAC’s) – a “fake”
chromosome containing foreign DNA with the ability
to replicate and undergo mitosis – up to 3000kb
e. Bacterial Artificial Chromosomes (BAC’s) – a fake
bacterial chromosome – can hold 100-300 kb
Example of Plasmid
Advantages of Plasmids as Vectors
Replicate quickly
 Has an origin of replication so is copied
and passes to daughter cells
 Doesn’t need to enter genome to be
copied
 Easy to put DNA into plasmids
 Easy to put plasmids into bacteria
 Can incorporate selection factors to make
it easy to find bacteria containing gene of
interest
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Host
Can use animal cell, plant cell, yeast cell, or bacteria. Bacteria are the
easiest – can get DNA in to the cell easier and they replicate faster
a. Difficult to put DNA into eukaryotic cells (can
electroporate, inject the DNA, or attach the DNA to
metal beads and shoot through membrane with a
gun) – usually use retroviruses!
b. Although bacteria are easy to use, you can’t
always use them because they don’t have
mechanisms to cut out introns and don’t do posttranslational modifications – if it needs these
modifications to be functional, must use a eukaryotic
cell
Making Recombinant DNA:
How do you clone a gene and how do find
the cells that have your gene in them?
1. Cut out your gene
of interest and put it in
a vector
o
o
o
Digest the plasmid
and DNA of interest
with same restriction
enzyme (now have
compatible sticky
ends)
Some plasmids will
close up without gene
Some plasmids will
get the gene inserted
How to Clone a Gene Continued
2. Transforming Bacteria (putting the gene
of interest with the plasmid into the cell)
Artificial Transformation
o
o
o
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Need competent cells (in exponential growth)
Positive ions make cell membranes permeable
to DNA
Can also use electroporation, heat shock
Can inject the DNA
Gene Cloning Continued
3. Selecting cells that have the gene of
interest
Need to know if the gene is inserted into the
plasmid and if the plasmid is inserted into the
cell
A. To see if colony has the plasmid - Use a plasmid that
has antibiotic resistance gene – if plasmid is
inserted, it will grow on antibiotic (like ampr – and
grow on amp)
B.
To see if plasmid containing colonies have
plasmids with the gene of interest:
o
Use restriction enzymes that cuts in the middle of a color
gene like the β galactosidase gene – if inserted the
colonies will be white
Selection of cells with gene of
interest continued
o
o
Look for protein products by activity or
antibodies
Make probes (short pieces of DNA or RNA
that will hybridize (base pair) with the gene
of interest – must be radioactive or
fluorescent.
o
Transfer some cells from each colony to filter
paper, probe, and then match up colonies
Technology #2 – Creating a
Genomic Library
1.
2.
3.
Cut whole genome with restriction enzymes – makes
sticky ends
Cut plasmids with same restriction enzyme to make
matching sticky
Mix the 2 together to get a bunch of plasmids – each
with a piece of the genome
Problems with Genomic Libraries
1.
There are many random fragments
2.
Must find the correct gene out of all of the plasmids
3.
Contains introns that bacteria can’t transcribe
Making a Genomic Library
Use same
restriction enzyme
to cut genomic
DNA and plasmids
to make matching
sticky ends
Technology #3: Creating a
cDNA Library
1.
Create the cDNA
(complementary DNA)
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1.
Collect all of the mRNA from a
cell
Use the enzyme reverse
transcriptase (from retroviruses)
to copy the mRNA into ds DNA
Cut cDNA with restriction
enzymes, cut plasmids with
same r.e. and mix together
PCR – polymerase chain reaction
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Make millions of copies of a single piece of DNA
due to primers (must know some sequences
flanking the gene)
No need to isolate the gene first
DNA can be old and in very small quantities
Can use for crime detection if only have 1 cell or
a small sample
Can use to amplify a gene of interest before
making a library so there is a higher
concentration of that gene in the library
PCR
1.
2.
3.
4.
5.
6.
7.
8.
Make primers complementary to the ends of
the target sequence
Heat denature the DNA (960)
Cool DNA – primers stick (500)
Heat a little and let DNA polymerase copy the
ds DNA (720)
Heat denature again
Cool
Copy, repeat, repeat, repeat
30 cycle makes 200 million copies
RT PCR
AAAAA
RT TTTTT
AAAAA
RT
TTTTT
AAAAA RT
TTTTT
Oligo dT primer is
bound to mRNA
Reverse
transcriptase
(RT) copies first
cDNA strand
Reverse
transcriptase
digests and
displaces mRNA
and copies
second strand of
cDNA
Double
strand
cDNA
Conversion of mRNA to cDNA by Reverse Transcription
50º
A. Double
strand DNA
96º
B. Denature
50º
C. Anneal
primers
Taq
D. Polymerase
binds
72º
Taq
72º
Taq
Taq
Taq
E. Copy
strands
Taq
1
96º
First round
of cDNA
synthesis (4
strands)
2
3
4
F.
Denature
Electrophoresis – Separation of
molecules based on electrical
charge and size
Uses:
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Determine the size of a fragment
Purify plasmids
Identify genes through hybridization/ Diagnosis
of genetic disease
Sequencing of a gene
RFLP or VNTR analysis
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Paternity testing
Forensics
Chromosome mapping
DNA Electrophoresis
Cut up DNA with restriction enzymes
 Load solution of cut up DNA into a well of an
agarose gel (porous gel that acts as a sieve)
 Apply an electrical current to gel
 DNA is negatively charged so it moves to the
positive pole.
 Since the gel is
porous – the smaller
the piece of DNA –
the faster it moves
so it separates by size
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How to pour, load, and run a gel
Electrophoresis after restriction
digestion
TATCTGGAAGTGGTACC GGAATCTACCGG
TATCCGGAAGTGATACCGGAATCTACCGG
TATCCGGAAGTGGTATCGGAATCTACCGG
Plasmid Purification
What a cut vs. uncut plasmid
looks like
Circular forms of DNA migrate in agarose
distinctly differently from linear DNAs of the
same mass. Typically uncut plasmids will appear to
migrate more rapidly than the same plasmid when
linearized. Additionally, most preparations of uncut
plasmid contain at least two topologically-different
forms of DNA, corresponding to supercoiled forms
and nicked circles. The image to the right shows an
ethidium-stained gel with uncut plasmid in the left
lane and the same plasmid linearized at a single
site in the right lane.
Gene Identification
If the DNA is purified
Cut the DNA
 Run gel (DNA runs toward the + pole)
 Stain if have purified gene
CCG↓CGGTAGGAAC CCACGGTAGGAAC
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Gene Identification
Using Genomic DNA
Cut the DNA
 Run gel (DNA runs toward the + pole)
 If stained the gel – big smear of DNA because
so many bands
 Southern Blot and Probe
 Probe = ATCCTT
CCG↓CGGTAGGAAC CCACGGTAGGAAC
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Southern Blotting
Denature DNA in gel
 Transfer to Nitrocellulose paper by
capillary action
 Probe with labeled probes
 Wash non-specific probe off of paper
 Expose to film
 Can see if a DNA sequence is there, how
many fragments, size of fragments
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Stain vs. Southern Blot with
Genomic DNA
Bacterial DNA Cut with a
Restriction Enzyme
Left is stained/ Right is Blotted and Probed
Other Blotting Techniques
Northern – same but using RNA instead of
DNA
 Western Blotting – electrical transfer of
proteins to paper and then using
fluorescently or radioactively tagged
antibodies to identify protein
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Alternative for Genetic Disease
Diagnosis
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Can diagnose diseases without gels using
RT-PCR
Collect cells – do PCR of a particular gene
 Sequence the gene to see if it is normal or
mutated
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Aside – can find presence of infectious
viruses this way – collect blood or cells
and PCR viral genes – run on gel or
sequence to see if present
RFLP or STR – Paternity Testing
and DNA Fingerprinting
RFLP – restriction fragment length polymorphisms
 Same as a Southern Blot but usually
probe for non-coding regions of DNA that
are highly variable (usually these regions
are more variable than actual genes)
 Usually use multiple probes
 STR (short tandem repeats)
 Found to be the most variable among
humans – makes fragments of different
sizes if different number of repeats in
satellite DNA
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DNA Fingerprinting
Must do stats for each probe – what is the % of
people that carry each restriction pattern for that
probe
The more probes, the more sure you can be that
the pattern fits only one person
6 probes is very good
Probe 1 – 1/10 exhibit this pattern
Probe 2 – 1/20
Probe 3 – 1/100
Probe 4 – 1/10
Probe 5 – 1/50
Probe 6 – 1/5
What is the chance that this profile can belong to
another person?
1/50,000,000
Combined DNA Index
System
Run by the FBI
Has over 5 million convicted
offender DNA profiles
Mandatory to have DNA profile in
CODIS if involved in a homicide or
sex crime
Uses 13 different loci to look at 13
different areas for differences in
their STR
Creates a unique pattern – 1 in 10
billion have matching pattern
Paternity Testing
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Run standard RFLP analysis
using 3-4 probes
The child gets 2 copies of every
gene – 1 from each parent
Every allele the child has, must
come from the mother or father
Alleged fathers can be excluded
but many times if the pattern
matches – there is only a 99%
chance that he is the father
Usually about 1/100 people
have that same pattern
Sequencing – Sanger Method
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Cut DNA with restriction enzymes
Divide into 4 reaction tubes with all the stuff for
replication – (DNA polymerase, ligase,
triphosphate nucleotides)
Add ddATP to 1 tube, ddTTP to 1 tube, ddGTP
to 1 tube, ddCTP to another tube
As DNA copies, eventually each nucleotide will
be “labeled”
Longest fragments at top, smallest at bottom,
read from the bottom up
Can do in one tube with fluorescently labeled
ddnucleotides – A,T,G,C a different color
Instruments for Sequencing
New Instruments
Microarray Analysis
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Gives the ability to compare gene expression between:
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Different tissues
Different species
Different stages of development
Cancer vs. non-cancer
Healthy vs. diseased tissue
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Procedure:
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Make the microarray plate (a robot attaches single stranded
pieces of DNA to a glass plate including every gene in the
genome)
Make cDNA from all mRNA in each of the two cell types you
want to compare
Make the cDNA ss and attach a red tag to the cDNA from one
cell type and a green tag to the cDNA from the other cell type
Add the tagged cDNA’s to the plate and let them hybridize
If the spot on the plate is red – it is only expressed in that cell
type, if it is green – only expressed in the other cell type, yellow
– expressed in both (can even tell concentration differences)
Affix ss genes to
glass plate
Represents all
genes of genome
A scanner reads the
amount of red
fluorescence and
green fluorescence
separately so you can
even tell slight
differences in gene
expression between
the two samples
In-Vitro Mutagenesis and
Transgenics
Try to get a handle on the function of the
gene
 Can change a gene and put it in a cell and
see the effect on the cell
 Can mutate a gene in an embryonic cell
and have it try to develop and see what
happens in development or in adulthood
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Cloning of Organisms
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Destroy DNA in egg
Put DNA from an adult somatic cell into the egg
Put the egg into a female host uterus and
develop into a new organism
Most are messed up, develop diseases, or die
prematurely
Using DNA with epigenetics (methylation,
acetylation patterns) of an adult cell – not of an
embryo – affects gene expression!
Embryo farms – grow balls of cells that are
clones to use for stem cells to grow new parts –
ethical?????
Stem Cell Research
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Pluripotent embryonic stem cells can become
any kind of cell
Adult stem cells become a particular kind of cell
or one of a few kinds of cells
Working on taking normal differentiated cells
and turning them back into stem cells so can use
them to make different kinds of cells (ex. Skin
becomes stem cell becomes a neuron)
Ethical issues? – where are we getting them
from?
Found some stem cells in brain – can make
some new neurons!