ChromoQuant® -Theory and interpretation of data. QF-PCR Quantitative Fluorescent Polymerase Chain...
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Transcript of ChromoQuant® -Theory and interpretation of data. QF-PCR Quantitative Fluorescent Polymerase Chain...
ChromoQuant®
-Theory and interpretation of data
QF-PCRQuantitative Fluorescent Polymerase Chain Reaction
What is QF-PCR?
QF-PCR uses fluorescent labelled primers to amplify STR regions from DNA, by PCR.
Short tandem repeats (STR) are highly polymorphic sequences found in the human DNA. The amount of fluorescently labelled amplicons are measured after fragment length separation in capillary electrophoresis.
QF-PCR is a quantitative method that determines the presence of different alleles which means the determination of chromosomal copy number
Which leads to: Diagnosis of fetal chromosomal ploidy within a working day!
(4-5 hours)
ChromoQuant® QF-PCR work flow
1. DNA extraction from e.g. amniotic fluid 2. PCR amplification 3. Capillary Electrophoresis (ABI or MegaBACE systems)4. Export of data and Diagnosis
From amniotic fluid to diagnosis within a working day!
+ 0oC
Extract DNA Prepare for PCR PCR Capillary Export data Electrophoresis
20 min 5 min 2,5-3h 30 min 5 min
Diagnosis
Validated instruments and DNA polymerases
ChromoQuant is designed to function with Applied Biosystems/Life Technologies or MegaBACE Genetic Analyzers:
ABI 3100, ABI 3130, ABI 3730, ABI 310, or MegaBACE
Validated Taq polymerases to be used with ChromoQuant:
Hot Start: HotStar Taq polymerase, Qiagen #203203 Hot Start: True Start Taq polymerase, Fermentas #EP0612Go Taq polymerase, Promega (not Hot Start!) #M8305KAPA, KAPABiosystems (under validation)
ChromoQuant workflow PCR preparation
10 l
Thaw and add Taq DNA polymerase
Vortex and quick spin down
Take Mastermix to PCR tube
Add purified DNA
+ 0oC
15 l4,4 µl for 10 tests
Run PCR: 2,5-3h
Freezer
<-18oCAlternatively store as Ready-to-use master mixes
To PCR
PCR preparation: 5 min
The QF-PCR method:Possible genotypes – normal or trisomic sample
Homozygote - non informative (possible trisomy)
Normal Heterozygote - informative 1:1
Trisomy 1:1:1Trisomy 2:1
Each PCR fragment peak corresponds to a STR (short tandem repeat) e.g.: (-GATA-GATA-GATA-)n .
Each peak uniquely represents an allele (one of maternal and one of paternal origin).
Upon PCR;
A normal (heterozygote) sample will generate two peaks.
A trisomy will generate two or three peaks. The ratio of the peaks will lead to the diagnosis.
Normal peak ratios
Ex 1) Chromosome 21 Ex 2) Chromosome 18
Peak ratio 1:1 Normal sample Peak ratio n/a Normal sample
or possible trisomy sample
Non-informative
1:1
Trisomy peak ratios
Ex 1) Chromosome 21 Ex 2) Chromosome 21
Peak ratio 1:1:1 Trisomy sample Peak ratio 1:2 Trisomy
sample
1:1:1
Trisomy 21
Trisomy 13
XY Extra Marker kit – Male and female samples
Panels for GeneMapper
Panels for ChromoQuant kits can be downloaded on www.cybergene.com
New! Unique marker for identification of Turner syndrome (X0)
A new marker called TAF9B has been introduced into the ChromoQuant XY Extra marker kit
TAF9B is a non polymorphic and stable marker with sequences found in the TAF9B gene on chromosome X, as well as in its pseudogene on chromosome 3
The specific peak for TAF9B on chromosome 3 is 205 bp, and represents 2 alleles
The specific peak for TAF9B on chromosome X is 210 bp, and represents 2 alleles in a normal female (46,XX karyotype)
In Turner syndrome (45,X0 karyotype in a female), this marker will represent only 1 allele. The chromosome 3 specific peak can therefore be used as a reference peak when determining the number of X chromosomes present´by calculation the area or height ratio between the two peaks
Experimental data