Download DNA_fingerprinting

DNA Fingerprinting:
The DNA of every individual is different. Loci where the human genome differs from
individual to individual are called polymorphisms.
1.
Polymorphisms may be different alleles of a gene (different base-pair
sequences), such as the ABO human blood group multiple alleles.
2.
Some polymorphisms are genes that exist in variable copy number.
3.
Other polymorphisms are non-coding DNA that varies in both base-pair
sequence and copy number, e.g. tandem repeats.
Tandem repeats are short DNA sequences that are non-coding and repeat at specific
loci a variable number of times. Both the sequence and the copy-number of
these repeats vary from individual to individual. These are the polymorphisms
targeted by DNA fingerprinting. E.g. there is a region of DNA just beyond the
insulin gene on chromosome 11, consisting of 7 to 40 repeats, depending on the
individual. E.g. TCATTCATTCATTCATTCAT is a short tandem repeat (STR) of 4
base pairs that repeats 5 times at the locus shown.
Ten or more different loci containing similar variable number tandem repeats are
tested, ensuring that the odds of a coincidental match are less than one in a
billion or even one in a trillion. These tandem repeats are used since the whole
human genome would be too much to analyse!
Procedure of DNA Fingerprinting in a forensic setting:
1.
A DNA sample must be obtained from the crime scene, e.g. a sample of spilt
blood. If the sample is large enough then DNA can be purified in sufficient
quantities, if the sample is too small then PCR must be used to amplify the DNA
in the sample.
2.
The purified DNA is cut by restriction endonucleases at either side of several loci
containing variable number tandem repeats (VNTRs). If the DNA has been
amplified by PCR then rather than restrict the DNA, it is possible to use primers
for DNA either side of these VNTR loci to only amplify these portions of DNA.
3.
Obtain DNA from the victim and from the suspects by taking blood samples.
Restrict or amplify these samples to obtain the same VNTR loci in these people.
4.
Separate the various VNTR DNA sequences obtained from each person by gel
electrophoresis. Use markers – DNA fragments of known and decreasing length.
Also use a control – DNA that has been fingerprinted before, to make sure that
the markers are reliable. DNA from the technician may also be tested as an
internal control in case of DNA contamination.
5.
Compare the DNA fingerprints of each suspect with the DNA sample from the
crime scene. If DNA from one of the suspects exactly matches the sample DNA
then a match ahs been obtained.
DNA Fingerprinting Exercise:
Blood found at a murder scene is thought to be that of the attacker (there was a vicious
fight and the attacker did not got away unscathed!). We have three suspects. Carry out a
DNA fingerprint on blood samples from each suspect and on blood found at the scene.
We are going to use restriction endonucleases and for simplicity we will only test two loci
(we aren’t really bothered about catching the real murderer, just so long as we get a
conviction!). Solve the crime!
1. Restrict each DNA sample with the following restriction endonucleases:
ECORI
—G—A—A—T—T—C— and Hind III —A—A—G—C—T—T—
—C—T—T—A—A—G—
—T—T—C—G—A—A—
And calculate the fragment sizes in bp.
Suspect one has been done for you already:
29 bp fragment
-GGCTACTGGAAGCTTAATGTCGGTCGGTCGGTCGGTCGTTCGAATTGCATGGGCG-CCGATGACCTTCCAATTACAGCCAGCCAGCCAGCCAGCAAGCTTAACGTACCCGC-GGCTATGGAATTCGGGTATAGTATAGTATAGCCAAGCTTGCTATTGCCCTGAGGCC-CCGATACCTTAAGC CCATATCATATCATATCGG TTCGAACGATAACGGGACTCCGG-
26 bp fragment
Suspect 2:
-CTGGAAGCTTGGCCGGCCGGCCGGCCGGCCGGCCGGCCGGCCGGCCTTCGAAT-GACCTTCGAACCGGCCGGCCGGCCGGCCGGCCGGCCGGCCGGCCGGAAGCTTA-TTGAATTCATCGATTAATTAATTACCAAGCTTGCTATTGCCC TGAGGCCGCTATGCT-AACTTAAGTAGCTAATTAATTAATGGTTCGAACGATAACGGGACTCCGGCGATACGASuspect 3:
-ATTGATTGATGGCTACTGGAAGCTTAACTAACTAACTGCTTCGAATTGCATCGGCG-TAACTAACTACCGATGACCTTCGAATTGATTGATTGACGAAGCTTAACGTAGCCGC-TACCTTAAGCATACATACATACATACATACATACATACATACATACATACAATTCGAA-ATGGAATTCGTATGTATGTATGTATGTATGTATGTATGTATGTATGTATGTTAAGCTT-
2. Plot the position the fragments would take on the following electrophoretic gel. The
blood found at the scene has already been done for you:
Base Pairs
(markers)
Crime
scene
Suspect 1 Suspect 2 Suspect 3
50bp
40bp
30bp
20bp
10bp
3. Which suspect do you think committed the murder?
Uses of DNA fingerprinting:
•
To test whether or not parents carry a disease-causing mutation that may be
passed on to their offspring, e.g. sickle-cell disease, cystic fibrosis.
•
To determine the real father of a child (paternal testing).
•
To test crime suspects to see if their DNA matches that found at the crime scene.
Problems with DNA fingerprinting:
False Negatives: mistakenly declaring that there is no match.
False Positives: mistakenly declaring that there is a match. Is this an acceptable error?
Although the probability of two people coincidentally having matching DNA fingerprints
is extremely small, the process of obtaining a DNA fingerprint by gel
electrophoresis is prone to experimental error. The same DNA sample will never
give two identical gels!