Forensic Biology by Richard Li

1. Lecture 11: Variable-Number Tandem Repeats Profiling Forensic Biologyby Richard Li

2. Basics • Human genome is abundant in tandem repeats • Minisatellites- 1980 • Also called Variable-Number Tandem Repeats (VNTRs) • Repeat unit > 10 bp (definition) • Often dozens to hundreds of bp per repeat • Genotype is defined by a particular number of tandem repeats at a given locus • Some have many alleles (possible numbers of repeats)

3. VNTR Loci • For forensics, VNTRs located far apart on the same chromosome or on different chromosomes (unlinked) • Review of independent assortment and behavior of unlinked genes • Review of rules of probability • Product Rule • Addition Rule • Examples

4. VNTR Loci • Population Match Probability (Pm) or RPM • Mathematical probability that two randomly chosen individuals will share the same genetic profile • The Lower Pm, the less likely a match will occur between two randomly chosen people • Pms as low as 10⁻¹² (1 in a trillion) have been calculated with VNTRs • Typically, run about 10⁻9 (1 in a billion)

5. Detection: Restriction Fragment-Length Polymorphism (RFLP) • Steps: • Extract DNA from sample • Digest DNA with restriction endonucleases • Separate fragments on an agarose gel • Denature DNA in the gel (single-stranded) • Transfer DNA to a nitrocellulose or nylon membrane (binds tightly to ssDNA) • Hybridize membrane with radioactively-labeled locus-specific ssDNA probes • Detect VNTR lengths by autoradiography

6. Detection: Restriction Fragment-Length Polymorphism (RFLP) 1. Extract DNA from sample • Can use any of the methods discussed in lab • For RFLP, there must be: • At least 50 ng of DNA (about 10,000 cells) • RFLP cannot be used to analyze trace evidence • Blood drop about the size of a nickel • A fresh ejaculate • DNA must be good quality (not degraded) • RFLP cannot be used on old/degraded samples (old bones) • Since the majority of forensic cases involve trace or degraded DNA, RFLP could only be applied in a small fraction of cases

7. Detection: Restriction Fragment-Length Polymorphism (RFLP) 2. Digest DNA with restriction endonucleases • Exonucleases versus endonucleases • Extracted from bacteria • Primitive immune system • Typically recognize palindromic sequences • E.g. 5’ – ACGT-3’ 3’ – TGCA – 5’ • Each restriction enzyme has its own site • Calculate # sites per genome • Calculate average size of fragments in a genome

8. VNTR locus D2S44; Each repeat unit is 31 bp in length Hae III = a restriction enzyme with a 4 bp palindromic recognition site

10. Hae III DNA digestion of human genome; fragments differ in length, with an average size of 4,096 bp.

11. Detection: Restriction Fragment-Length Polymorphism (RFLP) 3. Separate fragments by agarose gel electrophoresis • Digest loaded onto well on gel • Electrophoresis separates fragments by size • For a Hae III digestion, >12 million bands • If stained with ethidium bromide, would appear as a smear; discrete bands cannot be seen • Some bands larger, some smaller, by random chance • Average band size = 256 bp

13. Detection: Restriction Fragment-Length Polymorphism (RFLP) 4. Denature DNA in the gel • Soak gel in basic solution to denature strands • Strands are not available for hybridization with a radioactively labeled probe 5. Transfer the DNA to a nitrocellulose or nylon membrane • Denatured DNA will bind tightly to the membrane when cross-linked with UV light 6. Hybridize membrane with ss DNA probe

15. Radioactively labeled probe; hybridizes specifically to unique DNA flanking VNTR region within Hae III fragment

16. Only fragments recognized and bound by the probe will be detected after autoradiography (on X-ray film)

17. Restriction Fragment-Length Polymorphism (RFLP) • The denaturation, transfer, and probing steps are called “Southern Blotting” • Sir Edwin Southern, Mid-1970’s • Detection of denatured DNA restriction enzyme digest fragments with labeled ssDNA probes • Later, “Northern blotting” was invented • Detection of RNA transcripts by labeled ssDNA or RNA probes • Followed by “Western blotting” • Detection of proteins with labeled antibodies, DNA sequences (DNA binding proteins), RNAs (RNA binding proteins), or protein binding partners (heterodimers)

19. Restriction Fragment-Length Polymorphism (RFLP) • Factors affecting RFLP results: • DNA degradation • Partial restriction digestion • Star activity of restriction enzymes • Under some conditions (e.g. deviations in suggested temperature, pH of digestion) can cleave non-specifically • Point mutations • May abolish or introduce a new restriction enzyme site • Electrophoresis and Blotting Artifacts

20. Amplified Fragment Length Polymorphisms (AFLP) • Some VNTR loci have short alleles and are amenable to PCR amplification • D1S80: 14-42 repeat units • Requires less DNA and better with degraded samples • Amelogenin typing • Replaced with STR system in 1990’s • STRs even shorter and lots more of them