1. Chapter 17: Variable-Number Tandem Repeats Profiling

2.  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) 2

3.  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 3

4.  Population Match Probability (Pm)  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) 4

5.  Steps: 1. Extract DNA from sample 2. Digest DNA with restriction endonucleases 3. Separate fragments on an agarose gel 4. Denature DNA in the gel (single-stranded) 5. Transfer DNA to a nitrocellulose or nylon membrane (binds tightly to ssDNA) 6. Hybridize membrane with radioactively-labeled locus-specific ssDNA probes 7. Detect VNTR lengths by autoradiography 5

6. 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 6

7. 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 7

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

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

11. 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 11

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13. 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 13

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

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

17.  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) 17

18.  Two types of probes: Single locusMultiple locus 18 Radioactively labeled probe; hybridizes specifically to unique DNA flanking VNTR region within Hae III fragment Radioactively labeled probe; hybridizes to the repeat unit in the VNTR, which can be shared by more than one VNTR locus

19.  Single-locus probes (SLP) ▪ Detects only one VNTR locus at a time ▪ 1983-first used in criminal investigation in U.K. ▪ Lacked power  Multiple locus probes ▪ Detect multiple VNTR loci simultaneously; greater power ▪ Sir Alec Jeffreys- 1984: “DNA Fingerprinting” ▪ Very useful in paternity cases but not for mixed samples, degraded samples or limited quantities of DNA ▪ Possible findings: Inclusion (with statistics), exclusion, Inconclusive 19

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22.  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 22

23.  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 23

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