Forensic Biology by Richard Li

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Forensic Biologyby Richard Li

• Chapter 17 Variable-Number Tandem Repeats
  Profiling

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Basics

• Human genome is abundant in tandem repeats
• Minisatellites- 1980
• Also called Variable-Number Tandem Repeats
  (VNTRs)
• Repeat unit gt 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)

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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

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VNTR Loci

• 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)

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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

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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

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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

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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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Hae III DNA digestion of human genome fragments
differ in length, with an average size of 4,096
bp.
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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, gt12 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

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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

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Only fragments recognized and bound by the probe
will be detected after autoradiography (on X-ray
film)
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Restriction Fragment-Length Polymorphism (RFLP)

• The denaturation, transfer, and probing steps are
  called Southern Blotting
• Sir Edwin Southern, Mid-1970s
• 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)

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Restriction Fragment-Length Polymorphism (RFLP)

• Two types of probes
• Single locus Multiple locus

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Restriction Fragment-Length Polymorphism (RFLP)

• 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

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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

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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 1990s
• STRs even shorter and lots more of them

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