Professional Documents
Culture Documents
VNTRs Stu
VNTRs Stu
In analyzing VNTR data, two basic genetic principles can be used:
Identity Matching- both VNTR alleles from a specific location must match. If
two samples are from the same individual, they must show the same allele pattern.
Inheritance Matching- the VNTR alleles must follow the rules of inheritance. In
matching an individual with his parents or children, a person must have an allele
that matches one from each parent. If the relationship is more distant, such as a
grandparent or sibling, then matches must be consistent with the degree of
relatedness.
RFLP
When DNA tests were first introduced in the late 1980’s,
Steps-
1. Isolation of DNA
2. Digestion of DNA with a restriction enzyme (micro-scissors)
3. Separation of DNA fragments on agarose gel
4. Transfer of DNA fragements from agarose gel to nitrocellulose
membrane
5. Hybridiation with radiolabelled probe
6. Visualisation of hybridised DNA with autoradiography
RFLP
The DNA is broken into fragments by cutting it with one of
several restriction enzymes.
These enzymes act as "molecular scissors," cutting the DNA
strand at specific, known sites, and producing shorter fragments
known as restriction fragments.
For example, the restriction enzyme HaeI cuts only at the
sequence "AGGCCA" (which occurs randomly about once every
4,000 base-pairs).
The restriction enzymes chosen for forensic RFLP tests cut in
areas that flank the VNTRs.
The goal of the test is to measure the length of these VNTR-
containing restriction fragments,
hence the overall procedure is called restriction fragment length
polymorphism analysis.
Restriction Endonucleases
Also called restriction enzymes
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
Few Restriction Enzymes
Target sequence
Enzyme Organism from which derived (cut at *)
5' -->3'
Bam HI Bacillus amyloliquefaciens G* G A T C C
Eco RI Escherichia coli RY 13 G* A A T T C
Hind III Haemophilus inflenzae Rd A* A G C T T
Mbo I Moraxella bovis *G A T C
Pst I Providencia stuartii CTGCA*G
Sma I Serratia marcescens CCC*GGG
Taq I Thermophilus aquaticus T*CGA
Xma I Xanthamonas malvacearum C*CCGGG
Restriction Endonucleases
Recognition sites have symmetry (palindromic)
5’-GGATCC-3’
Bam H1 site:
3’-CCTAGG-5’
Restriction Endonucleases
Enzymes recognize specific 4-8 bp sequences
EcoRI 5’…GAATTC…3’
3’…CTTAAG…5’
PvuII 5’…CAGCTG…3’
3’…GTCGAC…5’
Recognition Sequences
5’ GAATTC 3’
3’ CTTAAG 5’
(Read the same in the opposite direction (eg. madam, race car…)
Sticky End Cutters
Most restriction enzymes make staggered cuts
Staggered cuts produce single stranded “sticky-
ends”
DNA from different sources can be spliced easily
because of sticky-end overhangs.
5’ P
- OH -
3’
HindIII ’
5
-P
H-
3’ O
EcoRI
Blunt End Cutters
Some restriction enzymes cut DNA at opposite base
They leave blunt ended DNA fragments
These are called blunt end cutters
AluI
HaeIII
Advantages
Highly polymorphic and reproducible
Transfer
The RFLP method of creating DNA profiles-
Southern hybridization and visualization
After their trip through the gel, the double stranded DNA
fragments are chemically split into two strands,
separating their paired nitrogenous bases from each other
(A from T and C from G).
These fragments are then directly transferred from the gel
(which, like gelatin desserts, is difficult to handle and
preserve intact) onto a sheet of a paper-like substance
(usually either made of nylon or nitrocellulose) called a
“filter" or "membrane."
The separated DNA fragments are then permanently
attached to the filter either by exposure to ultraviolet light
or cross-linking chemicals.
The RFLP method of creating DNA profiles-
Hybridization
A restriction enzyme that recognizes a four nucleotide
long restriction site (like HaeIII mentioned above)
should find such a site once every 256 base pairs on
average if each of the nucleotides are equally represented
in a random sequence.
For a 3.2 billion nucleotide sequence such as that of the
human genome, cutting with such an enzyme results in
literally millions of fragments of a very wide variety of
sizes.
As a result, the gel electrophoresis of a restriction
enzyme digested human genome is best described as a
smear of fragments that contains no distinct bands.
The particular bands of interest to forensic scientists are recognized
through the use of "probes" that seek out and bond with a locus of
interest and no other.
The tendency for A's and T's to interact and for G's and C's to interact
allows single stranded DNA molecules to be designed that stick more
stably to complementary sequences of nucleotides attached to the
membrane of the Southern blotting step described above.
Such probes can either be made through the use of recombinant
technology, or chemically synthesized.
These probes were originally tagged with radioactive markers that made
it possible to determine where they had attached to a membrane but
safer and more convenient fluorescent markers are also now available.
Probes that do not find a complementary sequence to which they can
bind are simply washed away while those that do bind give rise to a bar
code type of pattern that is characteristic of the VNTR DNA typing
methodology.
First application of DNA profiling in
a criminal investigation.
SLP was used to analyze samples. A
= hair roots from the first victim. B
= mixture of semen and vaginal
fluid from first victim. C = blood
from second victim. D = vaginal
swab from second victim. E =
semen stain on clothing from
second victim. S = blood from
suspect. Alleles (arrows) are
matched with the profiles of the
two cases but not with the suspect
profile.
Classes of VNTRs
There are two principal families of VNTRs:
microsatellites and minisatellites.
The former are repeats of sequences less than about 5 base pairs in
length (an arbitrary cutoff ), while the latter involve longer blocks.
Confusing this distinction is the recent use of the terms Short
Tandem Repeat (STR) and Simple Sequence Repeat (SSR), which are
more descriptive, but whose definitions are similar to that of
microsatellites.
VNTRs with very short repeat blocks may be unstable - dinucleotide
repeats may vary from one tissue to another within an individual,
while trinucleotide repeats have been found to vary from one
generation to another.
The 13 assays used in the CODIS database are usually referred to as
STRs, and most analyze VNTRs that involve repeats of 4 base pairs.
Summary
VNTR alleles are hypervariable regions of human
DNA that differ from each other in:
A. location of internal sites recognized by restriction
enzymes.
B. variable number of point mutations.
C. number of copies of an internally repeated DNA
sequence.
D. variable location on different chromosomes.
E. ability to hybridize with a specific single locus probe.
Introduction
STRs are currently the most commonly analysed genetic polymorphism in forensic
genetics. They were introduced into casework in the mid-1990s and are now the
main tool for just about every forensic laboratory in the world – the vast majority
of forensic genetic casework involves the analysis of STR polymorphisms.
A short tandem repeat (STR) in DNA occurs when a pattern of two or more
nucleotides are repeated and the repeated sequences are directly adjacent to each
other. The pattern can range in length from 2 to 10 base pairs (bp) (for example
(CATG)n in a genomic region) and is typically in the non-coding intron The
number of repetitions varies from one person to another and a particular number
of repetitions is known as an allele of the marker.
A short tandem repeat polymorphism (STRP) occurs when homologous STR
loci differ in the number of repeats between individuals. By identifying repeats of
a specific sequence at specific locations in the genome, it is possible to create a
genetic profile of an individual. There are currently over 10,000 published STR
sequences in the human genome. STR analysis has become the prevalent analysis
method for determining genetic profiles in forensic cases.
They are scattered through out the genome and occur on average every 10,000
nucleotides.
Analysis and types of STR
Determine the flanking regions surrounding the repeats.
Design PCR primers, amplify the repeat region.
New STR markers are identified in 2 ways-
Search DNA sequence data bases for regions with more
than 6 or so contiguous repeat units, or
Performing molecular biology isolation methods.
The STR repeat sequences are named by the length of the
repeat unit. They are-
dinucleotides, trinucleotides, tetranucleotides,
pentanucleotides and hexanucleotides.
Tetranucleotide repeats are the most popular STR
markers for human identification.
Statistical Analysis of DNA
Simple Repeats
Identical length and sequence
agat agat agat agat agat
Compound Repeats
Two or more adjacent simple repeats
agat agat agat ttaa ttaa ttaa
Complex Repeats
Variable unit length & possible intervening seq
agat agat aggat agat agat ttaacggccat agat agat
STR LOCI ALLELES
TPOX
THYROID PEROXIDASE
Chromosome 2
AATG repeat
6 to 13 repeats
TH01
TYROSINE HYDROXYLASE
Chromosome 11
TCTA repeat (Bottom strand)
4 to 11 repeats
Common microvariant 9.3
STR LOCI ALLELES
vWA
von Willebrand Factor
Chromosome 12
TCTA with TCTG repeat
10 to 22 repeats
D3S1358
Chromosome 3
AGAT with AGAC repeat
12 to 20 repeats
13 CODIS Core STR Loci
with Chromosomal
Positions
TPOX
D3S1358
TH01
D8S1179
D5S818 VWA
FGA D7S820
CSF1PO
AMEL
D13S317
D16S539 D18S51 D21S11 AMEL
Forensic DNA Analysis
PCR
Extraction
All DNA
Hundreds of
copies
PCR
16 Regions of DNA
10 million copies
This is what we want to
Run on Genetic Analyzer
do this week.
Short Tandem Repeats > Capillary Electrophoresis
12
Fluorescent Light
Laser
DNA
Short Tandem Repeats > Capillary Electrophoresis
12
194
206
186 7
190 8
194 9 194 9
198 10
202 11
206 12 206 12
210 13
From TPOX
9
12
From vWA
10
11
9 From TPOX
12
From vWA
10
11
210 13
202 11 202 ?
206 12
210 13
206 12 206 ?
210 13
From TPOX
9
12
From vWA
10
11
From TPOX
9
12
From vWA
10
11
9 From TPOX
12
From vWA
10
11
12
From vWA
10
11
210 13
202 11 202 11
206 12
210 13
From TPOX
9
12
From vWA
10
11
From TPOX
9
12
From vWA
10
11
210 13
vWA vWA
232 ?
236 ?
210 13
236 11 236 11
240 12
244 13
D5S818 D13S317
D7S820
ALLELIC LADDERS
PCR Product Size (bp) Same DNA Sample Run with Each of
the ABI STR Kits
D3S1358 vWA FGA
Power of Discrimination
Blue 1:5000
...C C A T T G A C...
…G G T A A C T G...
...C C G T T G A C...
…G G C A A C T G...
Identification of SNPs has been one of the most significant
outcomes of the Human Genome Project.
They are found in the human genome about once in every
1000 bp. It has been estimated that > 1 million common
SNPs, each with a frequency of 10% - 50% account for the
bulk of human DNA sequence difference. This represents
about 85% of the human genetic variation.
They are an important part of the future of differentiating
between individuals. A no. of technologies are being
developed to miniaturize and automate the procedure for
SNA analysis.
Single nucleotide polymorphisms may fall within
Types of SNPs