Lecture 10 Flashcards
in what situation do we use a cytogenic approaches instead of molecular approaches?
if we have very big alterations, insertions or deletions of different mega bases at the chromosomal level
what do we used of there are only small insertions or deletions of a few megabases or if there is the lacking of one entire exon?
classical PCR
what can the intrinsic bias of classical PCR cause?
allele dropout → during the amplification exploiting the classical PCR, there is the design two different primers (forward and reverse), and after the annealing there is
the elongation and the copy of the target of interest: if there is an alteration, for example a heterozygous deletion, there will be the annealing of only one primer and not the complete amplification, resulting in the amplification of only one allele, while the other one will be not amplified → we are not able to detect that lack of amplification
what are the three approaches used for the identification of insertions or deletions at the gene level?
- multiplex PCR of short fluorescent fragments (MPSF)
- multiplex amplification and probe hybridization (MAPH)
- multiplex ligation-dependent probe amplification (MLPA)
what has MPSF been developed to detect?
large insertions and deletions on two genes (MLH1 and MLH2) which are mismatch reaping genes associated with hereditary non-polyposis colorectal cancer
describe MPSF:
a quantitative method based on the multiplex amplification of all exons composing the specific gene exploiting probes labelled with fluorescence → we amplify different regions of the gene in the same run or analysis exploiting the use of fluorescent fragments
how many primers are used for MPSF?
two, in order to reduce the difference occurring during the amplification and to have the same amplification for all regions the gene in the same run
what is mandatory in MPSF analysis?
to have a wild type as a control analyzed in the same run and at the same time as out samples
what does it mean if the peak of the sample is higher than the wild type?
there’s a duplication
what does it mean if the peak of the sample is lower than the peak of the wild type?
there is a deletion
what type of deletion occurs if the reduction of the peak area is 0.5?
a heterozygous deletion
what does it mean if the increase in peak area is 1.5?
there is a heterozygous duplication
STUDY EXAMPLE FOR MPSF
pg 2-3 sbobine
in order to exploit the MPSF approach, with is required?
a large amount of starting DNA → also important to extract all that DNA in the exact same way to avid contamination due to different approaches of extraction
what type of approach is MPSF and what is the main idea?
quantitative → comparison of the sample with the control based on multiplex amplification of different regions of interest
what was QMPSF created for?
modified version of MPSF for the analysis of particular kinds of genes:
- genes characterized by the presence of GC regions (difficult to amplify)
- for pseudogenes (regions of he genome with high homology with the genes
how id QMPSF modified in order to find certain genes?
modified primer → the primer for the amplification presents an extension of 6 nucleotides in the 5’, a TAG, which is not complementary to the target region, but is only useful to have similar sizes of the amplicon, to have the same amplification at the same time of all the exons.
It is similar to the previous approach because the primers are labelled with fluorescence and there is
also the amplification of the wild type together with the sample. After the analysis on the sequencer, we obtain different peaks with different colours in the wild type and in the mutated sample, and it is possible to compare the area of each peak
REVIEW QMPFS
pg 4-5
with QMPFS, can we confirm the result, because with a a deletion we are sure that the primers were designed very far away, so are you able to create a fragment?
yes : you know where the deletion is so you can design the primers in the adjacent region
in QMPFS, is there a specific reason why the exons are not in order in the graphical representation?
it depends on the feature of the region and on the amplification condition of different exons - in this case you don’t have one single multiplex, but it is necessary to have four different multiplexes for the BRCA1 because it is a difficult gene to amplify using PCR
