Chapters 8, 9, 10 - Recombinant DNA Technology and Other Molecular Methods Used in Genomic Analysis Topics to be covered

1. Chapters 8, 9, 10 - Recombinant DNA Technology and Other Molecular Methods Used in Genomic Analysis • Topics to be covered today: • DNA Fingerprinting (DNA Typing/Genetic Profiling) • Application of restriction enzymes to study patterns of genetic polymorphism (RFLPs) • Southern Blots & Northern Blots

2. 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. • Mutation (including insertion & deletion) • DNA replication errors (same effect as mutation) • These processes produce variation in both DNA sequence composition and fragment size.

3. DNA Fingerprinting (DNA typing/profiling) Some Types of markers: • RFLPs (Restriction Fragment Length Polymorphisms) – a way of detecting polymorphism using restriction enzymes + PCR • Allele specific oligonucleotide probes that amplify known fragments of different sizes • Repeated DNA • 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’

4. Fig. 9.1 2nd edition, minisatellite repeat (VNTR) – i.e., Jeffreys Probes

5. Four criteria for selecting useful DNA fingerprinting markers: • Markers should be polymorphic. (so that they are variable and informative) • Markers should be single locus. (so that they occur in only one location in the genome and there is no ambiguity about their number or position) • Markers should be located on different chromosomes. (so that the markers are independent and randomly distributed) • 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)

6. 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. • If DNA misaligns and loops out on new strand, fragment size increases. • If DNA misaligns and loops out on template, the fragment size decreases. • This most frequently occurs by a multiple of the microsatellite repeat. Ellegren (2004)

7. Microsatellites are excellent marker for DNA fingerprinting because: • Polymorphic - fast-evolving due DNA slippage during DNA replication. • Single locus – they are assayed by PCR using 2 primers that bind to know points in the genome. • Common and throughout genomes of most organisms. • 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

8. How to fingerprint alleged paternity using microsatellites: Extract DNA from mother, baby, and alleged father. Synthesize oligonucleotide microsatellite primers and label one primer with fluorescent dye (2 primers per microsatellite). Amplify microsatellites using PCR from mother, baby, father. Electrophores microsatellite PCR products on a DNA sequencer (w/polyacrylamide) with a flourescent size standard loaded in the same lane or capillary. 3-4 different microsatellites can be multiplexed in each lane or capillary by using 3-4 different fluorescent dyes. 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). Sequence at least one copy of each allele to verify allele sizes.

9. Size Mother Baby “Father” Hypothetical gel pattern for microsatellite heterozygous for all individuals.

10. Paternity Analyses & Conclusions: • Baby and mother are expected to share on allele, and the baby and father the other allele. • If baby and father do not share a common allele, the “father” is not the father. • 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. • 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. • To achieve high probability, 6-12 loci should be assayed (exact number depends on variation in population for each marker). • If each locus has few alleles, more loci are required. If allelic diversity if high, fewer loci can be analyzed.

11. 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.

12. Analysis of Restriction Enzyme Sites: Restriction enzymes are useful tools that can be used to generate recombinant DNA and recombinant DNA libraries. Genomes vary naturally in the number and distribution of particular restriction sites. 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

13. Analysis of Restriction Enzyme Sites: The number and positions of restriction sites can be used for Dna fingerprinting just like microsatellite. The sequence of particular sites on a chromosome can be used to generate a linkage map that can have many applications: e.g., forensics, systematics, population genetics. DNA is cut with different enzymes, and each DNA-enzyme mixture is loaded on a separate lane in agarose gel. Negatively charged DNAs separate by size in the electric field (smaller fragments move faster than larger fragments). Fragment pattern produced by gel stained with ethidium bromide is photographed or imaged and processed by hand or using software. Distance each band migrates is calibrated with known size standards of pre-cut DNA (i.e., ladder). Resulting pattern of different numbers and sizes of fragments cut by different restriction enzymes is interpreted to make a restriction site map.

14. Large bands - migrate slower Small bands - migrate faster 0.5 2.5 2.0 EcoRI BamHI

16. USDA-ARS - RFLP (restriction fragment length polymorphism) analysis of PCR-amplified 16S rDNA sequences.

17. Applications of Restriction Enzyme Site Analysis: Analyze gene structure with large and fine scale mapping using a variety of different restriction enzymes. Can analyze a single gene using a PCR product (simplest), or analyze entire genome (more complicated) . The resulting fragment patterns are called Restriction Fragment Length Polymorphism (RFLPs) RFLP patterns 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. Less expensive that DNA sequencing/other genotyping methods. Another application is that 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 and how many copies of a gene might exist.

18. 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.

19. Fig. 8.8

20. Fig. 10.8, Southern Blot Components: (top to bottom) Weight Paper towels Membrane Gel Blotting paper Glass plate Tray with buffer

21. 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_pages/CNS/sybII.html