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Forensic Biology by Richard Li

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1 Forensic Biology by Richard Li
Lecture 11: Variable-Number Tandem Repeats Profiling Forensic Biology by 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 Transfer the DNA to a nitrocellulose or nylon membrane Denatured DNA will bind tightly to the membrane when cross-linked with UV light 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: 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


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