Lecture 11: Variable-Number Tandem Repeats Profiling.

Lecture 11: Variable-Number Tandem Repeats Profiling

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) 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)

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Steps:1. Extract DNA from sample2. Digest DNA with restriction

endonucleases3. Separate fragments on an agarose gel4. 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

labFor 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 systemTypically 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.

3. Separate fragments by agarose gel electrophoresis

Digest loaded onto well on gel Electrophoresis separates fragments by

sizeFor 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

strandsStrands are not available for hybridization

with a radioactively labeled probe

5. Transfer the DNA to a nitrocellulose or nylon membraneDenatured 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)

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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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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Some VNTR loci have short alleles and are amenable to PCR amplification

D1S80: 14-42 repeat unitsRequires less DNA and better with

degraded samples Amelogenin typingReplaced with STR system in 1990’s

STRs even shorter and lots more of them20

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