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!

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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

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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