1. -Theory and interpretation of data
ChromoQuant®
-Theory and interpretation of data

2. 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)

3. ChromoQuant® QF-PCR work flow
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!
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Extract DNA Prepare for PCR PCR Capillary Export data Electrophoresis
20 min min ,5-3h min 5 min
Diagnosis 3

4. 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)

5. ChromoQuant workflow PCR preparation
10 ml
Thaw and add Taq DNA polymerase
Vortex and quick spin down
Take Mastermix to PCR tube
Add purified DNA
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15 ml
4,4 µl for 10 tests
Run PCR: 2,5-3h
Freezer
<-18oC
Alternatively store as
Ready-to-use master mixes
To PCR
PCR preparation: 5 min

6. 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.
Homozygote - non informative (possible trisomy)
Normal Heterozygote - informative 1:1
Trisomy 1:1:1
Trisomy 2:1
Only heterozygote pattern can be used for correct interpretation . Shortest allele divided by longest allele
Trisomy peak ratios 2:1 (1:2)
>1,8 (<0,65)
Normal peak ratios: > 0,8 < 1,4

7. 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
1:1
Non-informative

8. Trisomy peak ratios 1:1:1 Ex 1) Chromosome 21 Ex 2) Chromosome 21
Peak ratio 1:1:1 Trisomy sample Peak ratio 1: Trisomy sample
1:1:1

9. Trisomy 21

10. Trisomy 13

11. XY Extra Marker kit – Male and female samples

12. Panels for GeneMapper
Panels for ChromoQuant kits can be downloaded on

13. 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

14. Experimental data
Sample 1: Normal male sample; For TAF9B: 2 alleles of chromosome 3, 1 allele of chromosome X; ratio 1:2. Single alleles for the other X markers. Prescence of chromosome Y-specific marker G10 STS47.
Sample 2: Turner female sample; For TAF9B: 2 alleles of chromosome 3, 1 allele of chromosome X; ratio 1:2. 1 allele for the other X markers. Absence of chromosome Y-specific marker G10 STS47.