Chapter 17: Variable-Number Tandem Repeats Profiling
Human genome is abundant in tandem repeats Minisatellites 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
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
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
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
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
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 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 Hae III DNA digestion of human genome; fragments differ in length, with an average size of 4,096 bp.
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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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 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)
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
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
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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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
Some VNTR loci have short alleles and are amenable to PCR amplification D1S80: 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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