Lecture 32 Flashcards
Why has PCR replaced RFLPs?
Because markers based on PCR are faster, cheaper and automatable with a higher throughput
What is the process of PCR?
Double stranded DNA is split by raising the temperature to 90
The temperature is lowered to about 60 to allow primers to anneal to each end of the target sequence
Taq polymerase extends the primers (up to 2kb)
The temperature may be raised slightly to speed this reaction
The temperature is then raised to 90 again to split the DNA allowing for another cycle to occur
Why can the long strands generated by PCR of target sequence plus flanking sequence be ignored?
Because this DNA will increase in a linear fashion while the target sequence will increase exponentially so after several cycles the concentrations of the long sequences are negligible
What are the characteristics of PCR
Simple, cheap, fast and automatable
Can amplify a small region
Is extremely sensitive so only a very small sample needs to be obtained, the downside of this means that contamination can be a large problem
Must use a thermostable polymerase
Length of reliably made DNA is 2kb although with different enzymes this can be extended to 20-30kb
What are the requirements of primer sequences for PCR?
Using the equation 4^(#ofbases in primer) a random 16-mer will only occur once per human genome so primers of >20bp will typically bind to only one region in the human genome
In most cases primers can not be exchanged between species
Must have and exact match for the last 4-5bp on the 3’ end of the DNA as a 3’ mismatch will block extension
If there is a mismatch in the middle of the sequence then a lower temperature must be used and 5’ mismatches are tolerated readily but may result in a product with extra bases
What are microsatellites?
Tandem repeats of 1-6 nucleotides which are interspersed frequently throughout the genome and are highly polymorphic between individuals
How are variable number tandem repeats generated?
They are generated by DNA polymerase slippage as there is a much higher error rate of the enzyme at these sequences
How can microsatellites be used?
They can be replicated via PCR and then have th segments run on a gel, due to the difference repeat numbers they will be different sizes which can be measured through a gel to gain information such as paternity, forensics etc
What is multiplexing?
When sets of primers for several different microsatellites are combined in one reaction with different fluorescent labels to be distinguished on one gel
What makes microsatellites effective markers?
They are highly polymorphic which when combined with multiplexing of 10-30 microsatellites there is a high power to distinguish individuals
How can microsatellites be used to predict disease?
Mostly outside coding regions so are not causal instead linkage is used to identify diseases, this can be increased in accuracy by using markers on both sides of the gene and using markers close to the gene
What is the difference between fully informative and partially informative information gained from microsatellites or RFLPs?
Fully informative data is when all alleles present are different
Partial data there are several people with the same allele making it more difficult to distinguish what is happening
What are SNPs and why are they useful?
Single nucleotide polymorphisms which are the newest genetic marker, they are capable of creating the most dense genetic maps and can be used with arrays to give huge amounts of throughput
What is the downside of SNPs compared to microsatellites?
SNPs are less polymorphic, with only 4 possible alleles and 2 common alleles so they have lower information content per SNP than microsatellite
What are the two ways of analysing SNPs through use of microarrays?
There are up to 1 million DNA spots in the microarray each with a specific sequence
In one way these can be analysed is to have four oligonucleotides per SNP (one for A,T,G and C) laser microscopy is then used to identify which one is the match as the complete hybrid while pair much better
The other method is to isolate the genomic DNA and to anneal olidogs with labelled dideoxynucleotides this is then denatured and the annealed to the microarray where the colour is scored to show the genotype
