DNA PROFILING
STR - CHARACTERISATION AND PRIMER DESIGN
Introduction
Successful PCR experimentation depends crucially on primer design and characterisation. Poor primer design can lead to a series of problems that can result in, for example, no amplification, poor amplification or the amplification of multiple targets.
There are some simple rules that can be used to predict suitable primers for the PCR:
• Primers obviously have to complementary to regions of the template DNA
• However, some other rules need to be followed
• Primers should be about 20 bases long
• G + C content should be about 45-55%
• Annealing temperatures should be within 1oC of each other (2oC for every A or T and 4oC for every G or C can be used)
• Primers should not form hairpins or anneal to each other
• Hairpins-primers can be “self-complementary”
5’ GTTGACTTGATATTCTCAAG 3’ can do:
5’ GTTGACTTGATA
T
3’ GAACTCT
• Primers should not anneal to each other
5’ ACCGGTAGCCACGAATTCGT 3’ –any potential problems??
5’ ACCGGTAGCCACGAATTCGT 3’
3’ TGCTTAAGCACCGATGGCCA 5’
You have been provided with the nucleotide sequence for
1. Homo sapiens tyrosine hydroxylase gene, which contains the TH01 STR
Relevant details of the STRs are as follows
TH01
Chromosomal location: 11p15.5 (tyrosine hydroxylase, 1st intron)
Repeat motif: TCAT (GenBank top strand) although AATG (bottom strand) often used. Note that variants with a [AATG]nATG[AATG]n structure quite common e.g. [AATG]6ATG[AATG]3 would be 9.3 repeats.
Allele range: 3-14
For the gene:
1. Identify and mark the introns and exons
2. Identify the repeat region
3. Identify the translation start codon
4. Identify the region where RNA polymerase binds
5. Calculate the number of repeats – does this fit with reported allele ranges?
6. Design suitable “forward” and “reverse” primers for the amplification of the repeat region
7. What would be the size in base pairs of the largest and smallest amplicons that would be expected if those primers were used in PCR.
(Remember that repeats are counted from the beginning of the first complete repeat to the end of the last complete repeat)
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