ChromoQuant®
-Theory and interpretation of data
QF-PCR
Quantitative 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.
2.
3.
4.
DNA extraction from e.g. amniotic fluid
PCR amplification
Capillary Electrophoresis (ABI or MegaBACE systems)
Export of data and Diagnosis
From amniotic fluid to diagnosis within a working day!
+ 0oC
Extract DNA
Prepare for PCR
PCR
20 min
5 min
2,5-3h
Diagnosis
Capillary
Electrophoresis
30 min
Export data
5 min
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 #EP0612
Go Taq polymerase, Promega (not Hot Start!) #M8305
KAPA, KAPABiosystems (under validation)
ChromoQuant workflow PCR preparation
Thaw and add Taq
DNA polymerase
Vortex and
quick spin
down
4,4 µl for 10 tests
Take Mastermix to PCR
tube
15 ml
Add purified DNA
Run PCR: 2,5-3h
10 ml
To PCR
+ 0o C
Freezer
PCR preparation: 5 min
Alternatively store as
Ready-to-use master mixes
<-18oC
The QF-PCR method:
Possible genotypes – normal or trisomic sample
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 Heterozygote informative 1:1
Trisomy 1:1:1
Homozygote - non
informative (possible trisomy)
Trisomy 2:1
Normal peak ratios
Ex 1) Chromosome 21
Ex 2) Chromosome 18
Peak ratio 1:1
Peak ratio n/a
Normal sample
or possible trisomy sample
1:1
Normal sample
Non-informative
Trisomy peak ratios
Ex 1) Chromosome 21
Ex 2) Chromosome 21
Peak ratio 1:1:1
Peak ratio 1:2
1:1:1
Trisomy sample
Trisomy sample
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