Chapter 17: Variable-Number Tandem
Repeats Profiling
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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)
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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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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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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
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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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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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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
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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
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Radioactively labeled probe;
hybridizes specifically to
unique DNA flanking VNTR
region within Hae III fragment
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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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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)
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Two types of probes:
Single locus
Radioactively labeled
probe; hybridizes
specifically to unique DNA
flanking VNTR region
within Hae III fragment
Multiple locus
Radioactively labeled probe;
hybridizes to the repeat unit in the
VNTR, which can be shared by more
than one VNTR locus
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 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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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 nonspecifically
 Point mutations
▪ May abolish or introduce a new restriction enzyme site
 Electrophoresis and Blotting Artifacts
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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
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