Lecture 11: Variable-Number Tandem Repeats Profiling
Dec 25, 2015
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)
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) 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)
4
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
5
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
6
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
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
9
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
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
11
12
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
13
14
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
18
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
19
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
21