-Theory and interpretation of data 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 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 Capillary Export data Electrophoresis 20 min 5 min 2,5-3h 30 min 5 min Diagnosis 3

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 10 ml Thaw and add Taq DNA polymerase Vortex and quick spin down Take Mastermix to PCR tube Add purified DNA + 0oC 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

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

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

Trisomy peak ratios 1:1:1 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 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.