DNA fingerprinting Flashcards
Applications of DNA fingerprinting
Immigration disputes
Paternity disputes
Murder cases
Identification
Length of a human chromosome
50-250 Mbp
How many chromosomes do humans have?
22 pairs of homologous chromosomes + 1 pair of sex chromosomes (XX = female, XY = male)
Therefore the human nuclear genome contains 23 pairs of chromosomes
Principle behind DNA fingerprinting
Although 99.9 % of human DNA sequences are the same in every individual, enough of the DNA is different that it is possible to distinguish one individual from another (unless they are identical twins) - this is their DNA fingerprint - each fingerprint is unique to that individual
These regions of variable DNA sequences between individuals are called ‘hypervariable regions’
Hypervariable regions are similar between closely related individuals (sequences are inherited from maternal/paternal chromosomes)
Hypervariable region
A region of DNA where a specific sequence is repeated in tandem
There are 2 types of these repeat sequences: microsatellites and minisatellites
Microsatellite
= Short Tandem Repeat (STR)
2-4 base pairs in length
Minisatellite
Variable Number of Tandem Repeats (VNTR)
Several 10s of base pairs in length
What is a single locus fingerprint?
A DNA fingerprint from just one hypervariable region on a chromosome
Hypervariable loci have…
…the possibility of mutations (they are unstable)
These mutations can be inherited
Makes it difficult to relate the fingerprint of a child to that of a grandparent
DNA profiling
New method of DNA fingerprinting Uses PCR instead of Southern blotting (which is much slower than PCR) Uses microsatellites (STRs) instead of minisatellites (VNTRs) Uses 16 pairs of primers for 16 different hypervariable loci (used to be 10 until 2014 - we are just looking at 10) The repeat unit at each hypervariable locus is 4 bp long (i.e. it is a microsatellite) e.g. 5' TCTA 3' Primers bind to either side of the hypervariable loci
Binding of primers in DNA profiling
The primers bind to the regions of DNA either side of the hypervariable locus
This is because the regions of DNA to which the primers bind are the same in both the maternal and paternal chromosome of an individual - only the hypervariable loci on each chromosome are different
i.e. so the forward and backward primers will amplify both the maternal and paternal chromosome, even though the hypervariable regions between them are different
Properties of primers for DNA profiling
One of the primers is labelled with a fluorescent tag (e.g. the forward primer is labelled)
(Only do one because it’s cheaper)
Labelled at 5’ end
4 pairs of primers employ FAM dye (blue)
3 pairs of primers employ JOE dye (green)
3 pairs of primers employ NED dye (yellow)
Altogether, 10 pairs of primers amplify all 10 hypervariable loci in one PCR (multiplex PCR) to give 20 products after one cycle
PLUS an additional pair of primers that amplify the amelogenin gene
Properties of dyes for DNA profiling
All aromatic
All highly conjugated
Therefore fluorescent
Amelogenin gene
Encodes a protein in tooth enamel
Amelogenin gene has 7 exons
A section of the intron between exons 1 and 2 is amplified
Present on both the X and Y chromosomes
On X, gene = 106 bp
On Y, gene = 112 bp
Therefore allows the sex of the individual to be identified
Process for obtaining a DNA profile
- Obtain DNA (e.g. via a swab)
- PCR
- Polyacrylamide gel electrophoresis (capillary electrophoresis)
Capillary electrophoresis
(Draw)
Capillary tube is filled with polyacrylamide gel - sorts DNA according to size
DNA is single stranded (unlike agarose gel electrophoresis) - DNA sample is in formamide, which outcompetes the DNA bases for hydrogen bonding
Blue light [from Ar laser (500 nm)] is shone at the end of the capillary tube - the fluorescent markers on the DNA strands fluoresce as they pass the laser
Why are peaks on a DNA profile in pairs?
One from maternal chromosome hypervariable region, one from paternal chromosome hypervariable region
Each pair of peaks corresponds to one hypervariable region
Why might only one peak be seen on a DNA profile instead of a pair?
That particular hypervariable region on the maternal and paternal chromosome are coincidentally the same length
Therefore the peaks they produce coalesce
How are the hypervariable regions used for DNA profiling chosen?
Carefully so that the peaks arising from each region do not overlap with those from the other regions
How many DNA profiles are in the UK national database?
~6 million
Why can male relatives be identified from their DNA profiles?
On the Y chromosome, there is a region of approx 150 Mbp containing 17 hypervariable loci
This region is stable over ~20 generations !
i.e. therefore every male in a family tree has the same Y haplotype
Y haplotype
Haplotype = a specific combination of repeats at each hypervariable locus
e.g. 12 repeats at locus 1, 11 repeats at locus 17 etc
Unlike other chromosomes, Y chromosomes do not come in pairs (except in males with XYY syndrome)
This means there is no chance variation of which copy of the Y chromosome is inherited, as well as no homologous recombination
Therefore, unlike with autosomal haplotypes, there is effectively no randomisation of the Y chromosome haplotype between generations
As a result, a human male should largely share the same Y chromosome as his father and so on
Restriction enzyme historically used in Southern blotting for DNA fingerprinting
Hinf1
Steps in DNA fingerprinting
- Isolate dsDNA
- Cut DNA using Hinf1 restriction enzyme to generate fragments for Southern blotting
- Run DNA on a gel (would have one lane of sample and one lane as a marker) to separate the fragments by size
- Blot DNA off gel onto a nylon membrane with layers of paper towels - DNA is immobilised onto the nylon membrane
- Pre-hybridisation: nylon membrane is washed with a prehybridisation solution containing salmon sperm DNA that helps to block non-specific DNA interactions and reduce background noise. The membrane is also washed in an alkaline solution to denature the DNA and give ssDNA
- Fold up nylon membrane and add to glass tube, alongside buffer solution and a radioactive oligonucleotide probe
- Hybridisation at approx. 60 degrees to ensure there is no non-specific binding of the probe to the DNA
- Tube is rotated to ensure the nylon membrane is fully covered in the probe solution
- Remove nylon membrane and add to light-proof box for several hours (box contains X-ray film)
- Will only be able to visualise DNA fragments on the X-ray film that had the correct base sequence to bind to the probe
How is DNA isolated from a cell for DNA fingerprinting?
- Cells are lysed - detergents/surfactants break down lipids from the cell/nuclear membranes
- Proteins are degraded by proteases
- RNA is degraded by RNases
- The solution is treated with saline to cause the broken protein/lipid/DNA debris to clump together
- The solution is centrifuged, which separates the DNA from the clumped cellular debris
- Any cellular/histone proteins still bound to the DNA can be removed by adding a protease or by precipitating the proteins with sodium/ammonium acetate
- DNA is then purified from the mixture using e.g. ice-cold ethanol/IPA - DNA is insoluble in these so will precipitate out
Oligonucleotide probe for Southern blotting
= oligonucleotide labelled at 5’ end with 32P
This labelling is done by T4 polynucleotide kinase, which adds this single 32P phosphate unit onto the 5’ end of ADP to generate gamma-32P ATP
(32P = beta emitter, t1/2 = 14 days)
What is the X-ray film called?
Autoradiograph
Southern blotting
= a method used in molecular biology for the detection of a specific DNA sequence in DNA samples
Probes used in Southern blotting in DNA fingerprinting application
Same sequence as the minisatellites
