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

Presence/absence of minisatellites are assayed by binding a complimentary oligonucleotide probe to repeat itself, not the flanking sequence.

ProbeCTCCGCC

Ellegren (2004)

Microsatellites (short tandem repeats or STRs):

Heterozygote Male 5’-TAATAATAATAATAATAATAA-------3’

Female 5’-TAATAATAATAATAATAATAATAATAA-3’

Homozygote Male 5’-TAATAATAATAATAA-3’(different allele) 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.

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 patternfor microsatellite heterozygousfor 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.

Uncut EcoRI BamHIEcoRI

+ BamHI

5.0 kb 4.5 kb 3.0 kb 2.5 kb

0.5 kb 2.0 kb 2.0 kb

0.5 kb

Small bands - migrate faster

Large bands - migrate slower

0.5 2.5 2.0

BamHIEcoRI

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)

WeightPaper towelsMembraneGelBlotting paperGlass plateTray 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 geneat different life stages in the frog Xenopus laevis

http://www.xenbase.org/WWW/Marker_pages/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