Chapters 8, 9, 10 - Recombinant DNA Technology and Other Molecular
Methods Used in Genomic Analysis
Topics to be covered today:
o
DNA Fingerprinting (DNA Typing/Genetic Profiling) with different
types of polymorphic markers
o
Application of restriction enzymes to study patterns of genetic
polymorphism (RFLPs)
o
Southern Blots & Northern Blots
DNA Fingerprinting (DNA typing/profiling)

No two individuals produced by sexually reproducing organisms
(except identical twins) have exactly the same genotype.
Why?

Crossing-over of chromosomes in meiosis prophase I.

Random alignment of maternal/paternal chromosomes in
meiosis metaphase I.

DNA replication errors & mutation
These processes produce variation in both DNA sequence
composition and fragment size.
Four criteria for selecting useful DNA fingerprinting markers:
1.
Markers should be polymorphic.
(so that they are variable and informative)
2.
Markers should be single locus/single copy.
(so that they occur in only one location in the genome and there
is no ambiguity about their copy number or position)
3.
Markers should be located on different chromosomes.
(so that the markers are independently distributed)
4.
For some applications such as the study of population size and
changes over time, markers should be neutral.
(so that they are not strongly affected selection; unless
selection is explicitly to be studied; selection confounds
estimates of population size parameters)
Some Types of markers used for DNA typing/profiling:

Two types of tepeated DNAs are most widely used.

Minisatellites (VNTRs = variable number tandem repeats)
Repeated units of 5 to several 10 bp – first discovered by A.
J. Jeffreys in 1985

Microsatellites (STRs = short tandem repeats)
Repeated units of 2-6 bp
5’-TAATAATAATAATAATAA-3’
3’-ATTATTATTATTATTATT-5’

RFLPs (Restriction Fragment Length Polymorphisms) – a way of
detecting polymorphism using restriction enzymes + PCR

Allele specific oligonucleotide probes/primers that PCR amplify
known fragments of different sizes
Fig. 9.1 2nd edition, minisatellite repeat (VNTR) – i.e., Jeffreys Probes
Probe
CTCCGCC
Presence/absence of minisatellites are assayed by binding a
complimentary oligonucleotide probe to repeat itself, not the
flanking sequence.
Microsatellites (short tandem repeats or STRs):
Heterozygote
Male
5’-TAATAATAATAATAATAATAA-------3’
Female 5’-TAATAATAATAATAATAATAATAATAA-3’
Homozygote
(different allele)
Male
5’-TAATAATAATAATAA-3’
Female 5’-TAATAATAATAATAA-3’

One proposed explanation for their fast rate of evolution is
slippage during DNA replication.
a)
If DNA misaligns and loops out on new strand, fragment size increases.
b)
If DNA misaligns and loops out on template, the fragment size decreases.
c)
This most frequently occurs by a multiple of the microsatellite repeat.
Ellegren (2004)
Microsatellites are excellent marker for DNA fingerprinting because:
1.
Polymorphic - fast-evolving due DNA slippage during DNA
replication.
1.
Single locus – they are assayed by PCR using 2 primers that bind
to know points in the genome.
1.
Common and throughout genomes of most organisms.
2.
Neutral – being non-coding repeated DNA they often have no
known function (though they might be linked to functional or
regulatory sequences).
http://www.brassica.info/resource/markers/ssr-exchange.php
Rates of extra-pair paternity in the human population are estimated to
range from 0.03% to 11.8%!
How to fingerprint alleged paternity using microsatellites:
1.
Extract DNA from mother, baby, and alleged father.
2.
Synthesize oligonucleotide microsatellite primers and label one
primer with fluorescent dye (2 primers per microsatellite).
3.
Amplify microsatellites using PCR from mother, baby, father.
4.
Electrophores microsatellite PCR products on a DNA sequencer
(w/polyacrylamide) with a flourescent size standard loaded in
the same lane or capillary.
5.
3-4 different microsatellites can be multiplexed in each lane or
capillary by using 3-4 different fluorescent dyes.
6.
Calculate size of each microsatellite relative to size standard
(this size standard also can be run in the same gel lane or
capillary using a 4th or 5th colored dye).
7.
Sequence at least one copy of each allele to verify allele sizes.
Size
Mother
Baby
“Father”
Hypothetical gel pattern
for microsatellite heterozygous
for all individuals.
Paternity Analyses & Conclusions:
1.
Baby and mother are expected to share on allele, and the baby and
father the other allele.
2.
If baby and father do not share a common allele, the “father” is not
the father.
3.
If the baby and father do share a common allele, paternity is
possible, but not proven, because other men in the population also
carry the allele at some frequency.
4.
Frequency of alleles that are shared in common by chance can be
calculated, and an appropriate number of microsatellites analyzed to
calculate probability of paternity.
5.
To achieve high probability, 6-12 loci should be assayed (exact
number depends on variation in population for each marker).
6.
If each locus has few alleles, more loci are required. If allelic
diversity if high, fewer loci can be analyzed.
Two examples using common and rare allelic variants:
Alleles are equally common (frequency = 0.25)
6 loci give probability of ID (PID) = (0.25)6 = 2/104
12 loci give PID = 6/108
Alleles are rare (frequency = 0.05)
6 loci give probability of ID (PID) = (0.05)6 = 2/108
12 loci give PID = 2/1016
The frequency of each allele in the population provides information
about the probability that allele is shared by chance.
This must be taken into account to determine appropriate
denominator/probability level to rule out false positive.
Analysis of Restriction Enzyme Sites:
1.
Restriction enzymes are useful tools that can be used to generate
recombinant DNA and recombinant DNA libraries.
2.
Genomes vary naturally in the number and distribution of
particular restriction sites.
3.
Cut patterns can therefore be used the map variation and study
relationships among individuals, within populations, and between
species.
http://lc-molecular.wikispaces.com/PCR+of+Target+Gene
Analysis of Restriction Enzyme Sites:
1.
The number and positions of restriction sites can be used for DNA
fingerprinting just like microsatellites.
2.
When DNA is cut, the sequence of particular sites on a
chromosome can be used to generate a linkage (restriction site)
map that can have many applications: e.g., forensics, systematics,
population genetics.
3.
The resulting fragment patterns are called Restriction Fragment
Length Polymorphism (RFLPs)
4.
Reflect the pattern of sequence variation within the genome
(presence/absence of particular polymorphisms). Each individual
possess a different pattern due to variation in number and
composition of different restriction sites.
5.
Less expensive that DNA sequencing/other genotyping methods.
Analysis of Restriction Enzyme Sites - Methods:
1.
DNA is cut with different enzymes, and each DNA-enzyme mixture
is loaded on a separate lane in agarose gel.
2.
Negatively charged DNAs separate by size in the electric field
(smaller fragments move faster than larger fragments).
3.
Fragment pattern produced by gel stained with ethidium bromide
is photographed or imaged and processed by hand or using
software.
4.
Distance each band migrates is calibrated with known size
standards of pre-cut DNA (i.e., ladder).
5.
Resulting pattern of different numbers and sizes of fragments cut
by different restriction enzymes is interpreted to make a
restriction site map.
EcoRI
+
BamHI
BamHI
Uncut
EcoRI
5.0 kb
4.5 kb
3.0 kb
2.5 kb
0.5 kb
2.0 kb
2.0 kb
Large bands - migrate slower
0.5 kb
Small bands - migrate faster
0.5
EcoRI
2.5
2.0
BamHI
USDA-ARS - RFLP (restriction fragment length polymorphism) analysis of PCR-amplified 16S rDNA sequences.
Other Important Application of Restriction Enzymes Site Analysis:
1.
Cut DNAs can be probed using the Southern Blot technique, which
can be used to determine if a gene shares homology with a probe
for a particular gene.
2.
Useful to determine how many copies of a gene might exist,
whether is single or multiple copy
Southern Blot:
•
Invented by Edward Southern.
•
After restriction enzyme cutting and electrophoresis, whole
genomic DNA appears as a continuous smear on gel.
•
Denature DNA in the gel with an alkaline solution.
•
Neutralize gel and place on blotting paper that spans a glass plate
and reaches into buffer solution.
•
Place a Hybridization membrane over the gel, and stack paper
towels and a weight on top of the membrane.
•
Buffer solution is wicked up by blotting paper to the paper towels,
passing through the gel and transferring DNA to membrane.
•
Fragments on the filter are arranged the same as on the gel.
•
Fix DNA in place on membrane by baking in hybridization oven.
•
Saturate the membrane with a homologous probe that binds
specific sequence and expose to x-ray film.
Fig. 8.8
Fig. 10.8, Southern Blot
Components:
(top to bottom)
Weight
Paper towels
Membrane
Gel
Blotting paper
Glass plate
Tray with buffer
http://www.biologyexams4u.com/2013/12/southern-blotting-principle-procedure.html
Northern Blot:
•
Similar to the Southern Blot, but used to probe RNA (not DNA).
•
Can be used to determine mRNA size, e.g., detect differences in
the promoter and terminator sites, etc.
•
Can be used to determine if a particular gene is expressed, and if
so, how much, what tissue type, and when in the life cycle?
Example:
Expression sybII gene
at different life stages in
the frog Xenopus laevis
http://www.xenbase.org/WWW/Marker_p
ages/CNS/sybII.html
Other types of Blotting Techniques:
•
Southern Blot – to probe cut DNA
•
Northern Blot – to probe RNA
•
Western Blot – to probe protein (protein immunoblot)
•
Eastern Blot – to analyze protein post-translational modifications
•
Southwestern Blot – to characterize DNA-binding proteins
•
Far-western Blot - to detect protein-protein interactions
•
Northwestern Blot – to detect protein-RNA interactions
•
Far-eastern Blot – analyis of lipids purified by HPLC
*Notheastern Blot & Southeastern Blot don’t exist, still to be developed