LECTURE 20 - MUTATIONS

what are the 2 main aspects of identifying variation in the genome

identification that region of DNA contains a variation

identification of a specific known DNA variation which has previously been identified

how do we detect heteroduplex

detected by product in gel electrophoresis

what does SSCPE stand for

single stranded conformational polymorphism electrophoresis

how does SSCPE work

bands occur in gel based on ssDNA conformation

how else can we detect heteroduplexes

denaturing gradient gel electrophoresis

denaturing HPLC

explain how a dot blot works

2 oligonucleotides are made; one complements the wild type gene, the other complements the mutant gene

oligonucleotides are labelled

oligonucleotides are hybridised at close to Tm

dot blot results

how does PCR-RFLP work

region is run through a PCR

the PCR product is digested with an RE
products are electrophroesed

resultant fragments depend on the DNA sequence variation present

what does ARMS stand for

amplification refractory mutational sequence

explain how ARMS works

2 primers are designed

each primer has a deliberate mismatch at the 2nd base from the 3’ end

wild type primer is the same as the wild type DNA sequence at the 3’ end, so it amplifies

mutant primer is the same as the mutant DNA sequence at the 3’ end, so it amplifies

double mismatches destabilise enough to prevent amplification

how can ARMS be done in 1 tube

we make 1 primer longer than the other

the type which receives the larger primer will appear at a shorter migration distance on the gel

explain mutational primer PCR

we design primers which mutates the region into a BAMHI site PCR
then we digest with BAMHI

the wild type digests, the mutant type does not

how do we produce a high resolution melt curve

we PCR the target sequence

an intercalating dye binds to dsDNA, and when bound it fluoresces brightly

this fluorescence is detected

therefore lots of detection at start of PCR process, then it decreases as dsDNA denatures

what does MALDI-TOF stand for

matrix assisted laser description ionization - time of flight

how does MALDI-TOF detect mutations

wild type and mutant type have different masses, and hence different times of flight

how can sequencing detect SNPs

the sequencing output is a graph like image, and SNPs can be read off of it