18.05.05 QF-PCR Flashcards
What is QF-PCR?
QF-PCR is a molecular method of determining the copy number of chromosome specific sequences by amplification of STRs on chromosomes of interest.
What are STRs? How are they utilitsed in QF-PCR?
STRs (short tandem repeats or microsatellites) are a pattern of two or more nucleotides that are repeated directly adjacent to each other.
Repeats can vary in length from 2 bp to 6 bp per repeat.
STRs are amplified by PCR using fluorescently labelled primers and separated by capillary electrophoresis. Repeat size and quantification.
STRs known highly polymorphic markers; a patient is therefore likely to have different numbers of repeat units on each allele.
What are the stages of QF-PCR set-up?
- DNA is extracted from the AF/CVS/blood/tissue
- Extracted DNA is added to the primer multiplex and PCR amplified over 24 cycles. This can be increased to 26 cycles in cases with small amounts of DNA. A
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PCR has 2 phases
- Exponential phase when all rx constituents are in abundance and amount of amplified product is directly proportional to original amount of starting material
- Plateau phase near the end of cycling, reagents are in short supply and some fragments may amplify and some may not. The results will be quantitative if PCR is stopped while in exponential phase i.e. after 26 cycles). - PCR products analysed on a capillary based genetic analyser alongside a size marker.
If there is a chance of overlap between the allele sizes of different markers these are labelled in different colours. - QF-PCR multiplex assays permit the detection of major numerical chromosome disorders within 1-2 days. Cheap to perform.
How many makers should be used per chromosome?
4
What is the composition of the majority of markers and PCR primers used in QF-PCR?
STRs are selected that exhibit high heterogeneity so that the copy number can be easily determined.
Majority of markers are usually 4bp repeats (tetra) but can also use tri/penta/hexanucleotide repeats. These have fewer stutter peaks (see below) than dinucleotide markers.
Dinucleotide repeat markers are acceptable if few suitable markers within the region of interest.
Markers should have high heterozygosity within the population to avoid uninformative tests.
Primers used must be >22bp in length and avoid homology to repetitive DNA and SNP’s.
What is the AMEL marker used for?
AMEL is a non-polymorphic marker with identical peaks in all patients, with a 4bp difference between the X and Y alleles. This cannot be used to quantify the number of X chromosomes in a female fetus, but can detect XXY,XYY
What is the TAF9 marker used for?
TAF9 is used to compare the number of X chromosomes relative to the number of chromosome 3s.
TAF9 primers amplify similar sequences present on on 3p24.2 and Xq21, which produce products 2bp different in length.
A normal female would be expected to have a 1:1 ratio (2 Xs and 2 3s), where as a normal male would be expected to have a 2:1 ratio of 3s:Xs (2 chromosome threes and 1 X).
What is the range for normal allele ratios?
Normal range for allele ratios should not exceed 0.8-1.4.
For alleles separated by more than 24bp, an allele ratio of 1.5 is acceptable as the smaller allele will be preferentially amplified.
How many informative makers are required for a normal interpretation of that chromosome?
Best Practice: Need at least 2 informative markers on a single chromosome with a normal biallelic pattern to interpret as normal (all other markers for that chromosome can be uninformative).
Can report a normal pattern with 1 marker but this must be stressed in the report.
What allele ratio is consistent with an abnormal triallelic pattern? What is the acceptable range?
1: 1:1
0. 8-1.4
What allele ratio is consistent with an abnormal biallelic pattern?
2: 1/1:2
0. 45 &0.65 and 1.8 &2.4.
How many informative makers are required for an abnormal interpretation of that chromosome?
Best Practice: Two informative abnormal markers required for trisomy (all other makers for that chromosome can be uninformative).
What can be interpreted about the mechanism of trisomy formation from the QF-PCR result?
The presence of 3 peaks for 1 or more marker is consistent with a meiosis I non-dysjunction event.
The absence of such a result (ie all abnormal markers have two allele with a 1:2/2:1 ratio) indicates a possible meiosis II or mitotic non-dysjunction event.
Where this pattern is seen in CVS the risk of CPM is increased (see Mosaicism).
What ratios indicate an uninformative result?
Ratios fall between normal and abnormal, i.e. 0.65-0.8 and 1.4/1.5-
Best Practice: Cannot report a sample as trisomic if any ratios are inconclusive or if any normal ratios for an otherwise trisomic chromosome are obtained.
Inconclusive ratios may be the result of preferential amplification of the smaller allele. This is more likely to occur if the distance between the alleles is increased, and may be more commonly observed for some markers than others.
How and why are trisomic results confirmed?
Confirm sample identity
In accordance with the current (2012) best practice guidelines, samples that give abnormal results should be confirmed before the result is issued (e.g. test maternal sample or second test on the original sample- according to individual lab policy).
