Introducing: 3Zomy Aneuploidity Test

Transcription

1 Introducing: 3Zomy Aneuploidity Test QF-PCR Life Technologies (India) Pvt. Ltd. 306, Aggarwal City Mall, Opposite M2K Pitampura, Delhi (INDIA). Ph: , , Mobile: Fax: Web: QF-PCR Tests for: Chromosome 13 Chromosome 18 Chromosome 21 XY

2 Prenatal Diagnostics Ultrasound and serum markers Laboratory diagnostics of chromosomal anomalies: QF-PCR Karyotyping FISH MLPA CGH Sequencing (Non Invasive)

3 What is QF-PCR? The quantitative fluorescent polymerase chain reaction (QF-PCR) has recently entered the field of prenatal diagnosis. The method allows rapid diagnosis

4 What is QF-PCR? This method tests the known chromosoms for aneupliodity to identify the most common trisomies in infants (trisomy 13, 18, 21) and to test the number of the sex chromosomes. QF-PCR is feasible on fewer cells.

5 Why QF-PCR? Results obtained within 24 hours from sample collect time The QF-PCR is a multiplex test, in a single reaction one can scan several mutations The QF PCR accuracy (over 98%) is a better solution than FISH It fully supports CGH results The protocol is user friendly

6 Overview of the procedure Clinical Sample (AF, CVS, blood or other tissue) DNA extraction PCR amplification of STRs markers Capillary electrophoresis (CE) Separation and identification of amplified DNA sequences Data interpretation Manual or Dedicated Software

7 Clinical Samples Amniotic Fluid (AF) Typically 1-3 ml is used for DNA extraction No need for culture Chorionic Villi Sample/Biopsy (CVS) - Two separate villi, 2-4 mm length, from different regions of the same sample should be analysed in parallell. Blood Less than 200 μl required Maternal blood sample should be analysed when Maternal Cell Contamination (MCC) is suspected in the prenatal sample Fetal blood, postnatal analysis

8 DNA extraction Selection of a high quality sample preparation method important assay performance and reproducibility fewer reruns instrument performance (capillary clogging) Measurement of DNA concentration sample archiving. Adsorption-desorption method recomended Silica column Magnetic beads Final DNA concentration Adjustment of elution volume based on pellet size Measuring DNA conc. Use Diluted TE or Water for DNA dissolving

9 PCR process 1. Starting DNA Template Temperature Ambient 2. Separate Strands (denature) 95 C 3. Add primers (anneal) 58 C 4. Copy (extend) 72 C Repeat cycle (steps 1-4 above) to copy DNA exponentially

10 Multiplex PCR + Simultaneous amplification of multiple loci in one reaction + Increases the amount of information obtained per reaction + Reduces amount of DNA template required - Primer design becomes more and more challenging as the level of multiplexing increases - Reaction optimisation is highly empirical and therefore time consuming - Stringent control of reagents and components necessary to guarantee reproducibility

11 Short Tandem repeats (STRs) The repeat region is variable between alleles (chromosomes) The flanking regions where PCR primers bind are constant The PCR product size is defined by the primer positions Terminology Homozygote: Both alleles are the same length and cannot be resolved Heterozygote: Alleles have different length and can be resolved

12 STR analysis normal genotype Allele 1 112bp 60 bp (15 x 4bp) 124bp 296bp Allele 2 112bp 36bp (9 x 4bp) 124bp 272bp Two allele copies - Heterozygous Allele 1 112bp 28bp (7 x 4bp) 124bp 264bp Allele 2 112bp 28bp (7 x 4bp) 124bp 264bp Two allele copies- Homozygous

13 STR analysis abnormal genotype Allele 1 112bp 48 bp (12 x 4bp) 124bp 284bp Allele 2 112bp 44 bp (11 x 4bp) 124bp 280bp 28bp (7 x 4bp) Allele 3 112bp Trisomic triallelic pattern 124bp 264bp Allele 1 72bp 60 bp (15 x 4bp) 72bp 204bp Allele 2 72bp 36bp (9 x 4bp) 72bp 180bp Allele 3 72bp 36bp (9 x 4bp) 72bp 180bp Trisomic diallelic pattern

14 Non-variable markers The amplicon size for one given chromosome is constant The amplicon size differs between different chromosomes Chromosomes are identified from the amplicon size using identical primers for the chromosomes tested Sex chromosome 202 bp Autosomal chromosome Symbols 180 bp Conserved regions Unique regions Labelled primer Unlabelled primer

15 Non-variable markers The following non-variable markers are utilized in our QF-PCR Kits: XY1 (AMELOGENIN: chromosome X / chromosome Y) Y1 (SRY: chromosome Y) TS7 (chromosome 7 / chromosome X) for Turner Syndrome TS3 (chromosome X / chromosome 3) for Turner Syndrome

16 QF-PCR: Interpretation Ratio Uninformative (1 allele) 4 bp Normal (2 alleles) 1:1 4 bp Trisomy (2 alleles, abnormal ratio) 2:1 4 bp 4 bp Trisomy (3 alleles) 1:1:1

17 Workflow Work-module (8 sample) Hands-On time Total time 1. PCR setup 2. DNA extraction 3. PCR amplification 4. Detection 5. Results analysis 10 min min 2 min 15 min 30 min 10 min 30 min 2 hrs 30 min 1 hr* Depending on the polymer 30 min Total < 1,5 hrs < 5 hrs*depending on the polymer

18 Product range QF-PCR 5 chromosomes 13, 18, 21, XY A mixed,one reaction markers, for all chromosomes together Trisomy Broad range Markers for chromosome 21 Sex Markers Broad range Markers for chromosomes XY

19 Kit Components PCR mix 1: PCR mix including oligonucleotides for amplification of markers on chromosomes 13, 18,21, X and Y PCR mix 21: PCR mix including oligonucleotides for amplification of markers on chromosome 21 PCR mix XY: PCR mix including oligonucleotides for amplification of markers on chromosomes X and Y PCR Enzyme: For preparation of final PCR mastermix when combined with the individual PCR mixes

20 Number of Tests, PCR mix 1: 2 vials, tests each PCR mix 21: 1 vial, tests PCR mix XY: 1 vial, tests PCR Enzyme: 4 vials,25-50 tests each

21 Matrix -Dye Set Capillary Electrophoresis (CE) detection calibration components needed for spectral calibration

22 Why Choose 3Zomy 3Zomy is being brought to you by Life Technologies (India) Pvt. Ltd. who has been a time tested & a vendor having good will in cytogenetics Market We have over a decade of experience in selling cytogenetics products (other tools) used in Karyotyping. Life Technologies (India) Pvt. Ltd. is associated with its principal partner Biological Industries who enjoy a distinctive leadership in cytogenetics Biological Industries is an exclusive supplier of several specialized human identity kits, tissue typing & trisomy detection with a team of STR specialists

23 3Zomy Advantages The kit can be used for: Full range trisomy Chromosome 21 full screening Sex chromosoms full screening Kit is a CE/IVD The results accuracy is over 98% The reaction volume can be optimised by customer even on the lower side (cost effective)

24 For Further Questions Contact Life Technologies (India) Pvt. Ltd. 306, Aggarwal City Mall, Opposite M2K Pitampura, Delhi (INDIA). Ph: , , Mobile: Fax: Web: