TITLE: Quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid prenatal diagnosis of common fetal aneuploidies
AUTHORS: Predrag Noveski, Marija Terzic, Marija Vujovic, Maja Kuzmanovska, Emilija Sukarova Stefanovska, Dijana Plaseska-Karanfilska

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">    Quantitative fluorescent polymerase chain reaction (QF-PCR) is reliable and efficient method for the rapid prenatal diagnosis of common fetal aneuploidies. In this protocol we first describe a procedure for isolation of DNA from amniocytes and chorionic villi with the use of commercial kit intended for the isolation of nucleic acids from viruses, which, taking into account the phenomenon of increased fragmentation of the DNA material of fetal origin, proved to be appropriate for prenatal diagnosis. (Section 1). As part of the isolation procedure, we also include a procedure for treating amniotic fluid with visible contamination (blood with possible origin from the mother).Тhe presented procedure consists of simple physical separation of the maternal blood cells from the fetal material in order to reduce the failure rate due to maternal cell contamination.</div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">    In the second part of the protocol (Section 2) we are presenting procedure for performing the QF-PCR analysis. Here, we describe an in-house one-tube multiplex QF-PCR method including 20 PCR markers (15 STR markers and 5 fixed size) for rapid prenatal diagnosis of chromosome 13, 18, 21, X and Y aneuploidies, which targeted in total 26 genomic positions. Of them, three markers (D13S258, D13S305 and D13S1817) were for analysis of the aneuploidies for the chromosome 13, four (D18S386, D18S390, D18S391 and D18S535)  for the chromosome 18, four (D21S1411, D21S1414, D21S1435 and D21S1446) for the chromosome 21 and six for the analysis of the sex chromosome aneuploidies. Two of the sex chromosome markers (DXS6803 and XHPRT) amplified sequences only on chromosome X (for counting chromosome X), two (AMELX/Y and DXYS218) co-amplified sequences of both X and Y chromosomes (for counting chromosomes X and Y in male samples and chromosome X in female samples), one (TAF9B) was for co-amplification of sequences on chromosomes X and 3 (for counting chromosome X) and one (SRY) located on chromosome Y was for male sex determination. Three remaining markers (MYPT2/Y, DYS448 and CDY1/2) were used simultaneously for detection of the Y chromosome aneuploidies and microaberrations in the azoospermia factor region “c” (AZFc), located on the Y chromosome. </div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">    For the cases with inconclusive results, we used additional QF-PCR reactions specific for given chromosome (Section 3). For chromosomes 13, 18 and 21 the primers were combined in one multiplex reaction each: four STR markers for chromosome 13 (D13S742, D13S628, D13S634 and D13S631), three STR markers for chromosome 18 (D18S51, D18S1367 and D18S978) and four  STR markers for chromosome 21 (D21S1437, D21S11, D21S1412 and D21S1441). For the sex chromosomes we have used three markers as separate PCR reactions (for DXS6809, DXS996 and X22) and additionaly if needed, previously published multiplex reaction [File 1] for detection of sex chromosome aneuploidies in infertile patients.</div><style>
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[STEPS]
?. [Main QF-PCR reaction]
Тhis section describes the main QF-PCR reaction and consists of the following steps:2.1) Prepare QF-PCR multiplex primer mix;2.2) Perform PCR reaction;2.3) Capillary electrophoresis of the PCR product and analysis of the results.
?. [DNA isolation from amniocytes/chorionic villi/blood]
Perfom one-hour DNA isolation protocol using the commercial High Pure Viral Nucleic Acid kit (Roche Applied Science, Mannheim, Germany):Prepare the material for isolation:For amniotic fluid -> in a 1.5 ml tube collect amniotic fluid, centrifuge 13000 x g for 5 minutes and remove the supernatant; repeat the procedure several times (depends on the amount of amniocytes and/or volume of amniotic fluid); after the last spinning, apart from the precipitate, also retain 200 ul supernatant; vortex and proceede with isolation according to the manufacturer's protocol.For chorionic villi -> in a 1.5 ml tube collect one or several chorionic villi and add up to 1.5 ml of 1x PBS buffer (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl; pH 7.4),   centrifuge at 13,000 x g for 5 minutes and remove the supernatant; repeat  the procedure one more time, discard the supernatant, add 200 μl 1x PBS and proceeded with isolation according to the manufacturer's protocol.For blood -> proceeded with isolation according to the manufacturer's protocol.Perform isolation with High Pure Viral Nucleic Acid kit according to the folowing procedure:
 Add 200 μl Binding bufferAdd 50 μl Proteinase K solution (supplied as apart of the kit); mix immediately. Incubate for 10 min at +72°C. Add 100 μl Binding Buffer and      mix well. To transfer the sample to a      High Pure Filter Tube: • Insert one High Pure Filter Tube into one Collection Tube. • Pipet the entire sample into the upper reservoir of the Filter Tube. • Insert the entire High Pure Filter Tube assembly into a standard table-top centrifuge.• Centrifuge 1 min at 8,000 × g. After centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. After combining the Filter Tube with a new Collection Tube: • Add 500 μl Inhibitor Removal Buffer (previosly prepared according to manufactutrer's description)  to the upper reservoir of the Filter Tube.• Centrifuge 1 min at 8,000 × g. After centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. After removal of inhibitors: • Add 450 μl Wash Buffer (previosly prepared according to manufactutrer's description) to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. After the first wash and centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. • Add 450 μl Wash Buffer to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. • Leave the Filter Tube-Collection Tube assembly in the centrifuge and spin it for 10 s at maximum speed (approx. 13,000 × g) to remove any residual Wash Buffer. The extra centrifugation time ensures removal of residual Wash Buffer. Discard the Collection Tube and      insert the Filter Tube into a nuclease-free, sterile 1.5 ml  microcentrifuge tube. To elute the nucleic acid: • Add 30 μl for amniocytes or 50 μl (chorionic villi or blood)of  Elution Buffer to the upper reservoir of the Filter Tube. • Centrifuge the tube assembly for 1 min at 8,000 × g. Dissolve the pelleted DNA with the pipetor and leave at 37 oC for one hour.Measure the DNA concentration and proceed to the QF-PCR reaction step.• Insert one High Pure Filter Tube into one Collection Tube. • Pipet the entire sample into the upper reservoir of the Filter Tube. • Insert the entire High Pure Filter Tube assembly into a standard table-top centrifuge.• Centrifuge 1 min at 8,000 × g. After centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. After combining the Filter Tube with a new Collection Tube: • Add 500 μl Inhibitor Removal Buffer (previosly prepared according to manufactutrer's description)  to the upper reservoir of the Filter Tube.• Centrifuge 1 min at 8,000 × g. After centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. After removal of inhibitors: • Add 450 μl Wash Buffer (previosly prepared according to manufactutrer's description) to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. After the first wash and centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. • Add 450 μl Wash Buffer to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. • Leave the Filter Tube-Collection Tube assembly in the centrifuge and spin it for 10 s at maximum speed (approx. 13,000 × g) to remove any residual Wash Buffer. The extra centrifugation time ensures removal of residual Wash Buffer. Discard the Collection Tube and      insert the Filter Tube into a nuclease-free, sterile 1.5 ml  microcentrifuge tube. To elute the nucleic acid: • Add 30 μl for amniocytes or 50 μl (chorionic villi or blood)of  Elution Buffer to the upper reservoir of the Filter Tube. • Centrifuge the tube assembly for 1 min at 8,000 × g. Dissolve the pelleted DNA with the pipetor and leave at 37 oC for one hour.Measure the DNA concentration and proceed to the QF-PCR reaction step.• Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. After combining the Filter Tube with a new Collection Tube: • Add 500 μl Inhibitor Removal Buffer (previosly prepared according to manufactutrer's description)  to the upper reservoir of the Filter Tube.• Centrifuge 1 min at 8,000 × g. After centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. After removal of inhibitors: • Add 450 μl Wash Buffer (previosly prepared according to manufactutrer's description) to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. After the first wash and centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. • Add 450 μl Wash Buffer to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. • Leave the Filter Tube-Collection Tube assembly in the centrifuge and spin it for 10 s at maximum speed (approx. 13,000 × g) to remove any residual Wash Buffer. The extra centrifugation time ensures removal of residual Wash Buffer. Discard the Collection Tube and      insert the Filter Tube into a nuclease-free, sterile 1.5 ml  microcentrifuge tube. To elute the nucleic acid: • Add 30 μl for amniocytes or 50 μl (chorionic villi or blood)of  Elution Buffer to the upper reservoir of the Filter Tube. • Centrifuge the tube assembly for 1 min at 8,000 × g. Dissolve the pelleted DNA with the pipetor and leave at 37 oC for one hour.Measure the DNA concentration and proceed to the QF-PCR reaction step.• Add 500 μl Inhibitor Removal Buffer (previosly prepared according to manufactutrer's description)  to the upper reservoir of the Filter Tube.• Centrifuge 1 min at 8,000 × g. After centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. After removal of inhibitors: • Add 450 μl Wash Buffer (previosly prepared according to manufactutrer's description) to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. After the first wash and centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. • Add 450 μl Wash Buffer to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. • Leave the Filter Tube-Collection Tube assembly in the centrifuge and spin it for 10 s at maximum speed (approx. 13,000 × g) to remove any residual Wash Buffer. The extra centrifugation time ensures removal of residual Wash Buffer. Discard the Collection Tube and      insert the Filter Tube into a nuclease-free, sterile 1.5 ml  microcentrifuge tube. To elute the nucleic acid: • Add 30 μl for amniocytes or 50 μl (chorionic villi or blood)of  Elution Buffer to the upper reservoir of the Filter Tube. • Centrifuge the tube assembly for 1 min at 8,000 × g. Dissolve the pelleted DNA with the pipetor and leave at 37 oC for one hour.Measure the DNA concentration and proceed to the QF-PCR reaction step.• Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. After removal of inhibitors: • Add 450 μl Wash Buffer (previosly prepared according to manufactutrer's description) to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. After the first wash and centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. • Add 450 μl Wash Buffer to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. • Leave the Filter Tube-Collection Tube assembly in the centrifuge and spin it for 10 s at maximum speed (approx. 13,000 × g) to remove any residual Wash Buffer. The extra centrifugation time ensures removal of residual Wash Buffer. Discard the Collection Tube and      insert the Filter Tube into a nuclease-free, sterile 1.5 ml  microcentrifuge tube. To elute the nucleic acid: • Add 30 μl for amniocytes or 50 μl (chorionic villi or blood)of  Elution Buffer to the upper reservoir of the Filter Tube. • Centrifuge the tube assembly for 1 min at 8,000 × g. Dissolve the pelleted DNA with the pipetor and leave at 37 oC for one hour.Measure the DNA concentration and proceed to the QF-PCR reaction step.• Add 450 μl Wash Buffer (previosly prepared according to manufactutrer's description) to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. After the first wash and centrifugation: • Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. • Add 450 μl Wash Buffer to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. • Leave the Filter Tube-Collection Tube assembly in the centrifuge and spin it for 10 s at maximum speed (approx. 13,000 × g) to remove any residual Wash Buffer. The extra centrifugation time ensures removal of residual Wash Buffer. Discard the Collection Tube and      insert the Filter Tube into a nuclease-free, sterile 1.5 ml  microcentrifuge tube. To elute the nucleic acid: • Add 30 μl for amniocytes or 50 μl (chorionic villi or blood)of  Elution Buffer to the upper reservoir of the Filter Tube. • Centrifuge the tube assembly for 1 min at 8,000 × g. Dissolve the pelleted DNA with the pipetor and leave at 37 oC for one hour.Measure the DNA concentration and proceed to the QF-PCR reaction step.• Remove the Filter Tube from the Collection Tube; discard the flowthrough and the Collection Tube. • Combine the Filter Tube with a new Collection Tube. • Add 450 μl Wash Buffer to the upper reservoir of the Filter Tube. • Centrifuge 1 min at 8,000 × g and discard the flowthrough. • Leave the Filter Tube-Collection Tube assembly in the centrifuge and spin it for 10 s at maximum speed (approx. 13,000 × g) to remove any residual Wash Buffer. The extra centrifugation time ensures removal of residual Wash Buffer. Discard the Collection Tube and      insert the Filter Tube into a nuclease-free, sterile 1.5 ml  microcentrifuge tube. To elute the nucleic acid: • Add 30 μl for amniocytes or 50 μl (chorionic villi or blood)of  Elution Buffer to the upper reservoir of the Filter Tube. • Centrifuge the tube assembly for 1 min at 8,000 × g. Dissolve the pelleted DNA with the pipetor and leave at 37 oC for one hour.Measure the DNA concentration and proceed to the QF-PCR reaction step.• Add 30 μl for amniocytes or 50 μl (chorionic villi or blood)of  Elution Buffer to the upper reservoir of the Filter Tube. • Centrifuge the tube assembly for 1 min at 8,000 × g. Dissolve the pelleted DNA with the pipetor and leave at 37 oC for one hour.Measure the DNA concentration and proceed to the QF-PCR reaction step.
?. [DNA isolation from amniocytes/chorionic villi/blood]
Treatment of maternal cell contamination in amniotic fluidsIn order to improve the outcome of the analysis of amniotic fluids defined as samples with maternal cell contamination (MCC), perform physical separation of the potentially contaminating material:Store the syringe with the amniotic fluid on +4oC for 6-12 hours in order to precipitate (precipitation could also be performed over night);After separation of the two clearly visible phases, firstly collect the upper clear phase in a 1.5 ml  tube without dissolving the lower phase; centrifuge at 11,000 x g for 5 minutes and discard the supernatant; repeat until all of the  upper clear phase is collected, and after last centrifugation retain 200 ul supernatant; After that, collect the lower  phase separately in a new 1.5 ml tube; centrifuge at 11,000 x g for 5 minutes and discard the supernatant, but retain 200 ul supernatant;After centrifugation of the collected amniotic fluid, perform separate isolation (as previosly described) and QF-PCR analysis for the two colected pellets . Visualisation of the procedure for the treatment of maternal cell contamination in amniotic fluids is shown in the following figure:
?. [Main QF-PCR reaction]
QF-PCR primer mix is prepared in a total volume of 200 μl.Sequences of the primers, their GC percentages, melting points and amount of each primer used in preparation of the QF-PCR mix are shown in the folowing table:  ABCDEFGH1NameSTR or fixed size (fs)Sequence 5 `-&gt; 3`GC %Tm 0CAmount (in μl) used in the primer mix from 50 pmol/μl  of Forward/Reverse primer stocksConcentration in the QF-PCR mix (nM)Final concentration in the QF-PCR
  reaction (nM)2AMEL-X/Y-Ffs6-FAM-CCC TGG GCT CTG TAA AGA ATA GTG50612500503AMEL-X/Y-RATC AGA GCT TAA ACT GGG AAG CTG46624DXS6803-FSTRVIC-GAA ATG TGC TTT GAC AGG AA40551537503755DXS6803-RCAA AAA GGG ACA TAT GCT ACT T36556DXYS218-FSTRPET-AAT CTG GGC CTG AAG CAT TT45581025002507DXYS218-RAAG AGC GAA ACT CCG TCT CA50608TAF9B-Ffs6-FAM-TTT GAC AGG TAG TTT TGG GTC A41583750759TAF9B-R1TGG TTT TGC CTA GGT CCA GT506010XHPRT-FSTR6-FAM-ATG CCA CAG ATA ATA CAC ATC CCC46617175017511XHPRT-RCTC TCC AGA ATA GTT AGA TGT AGG425612D13S258-FSTRNED-ACC TGC CAA ATT TTA CCA GG45578200020013D13S258-RGAC AGA GAG AGG GAA TAA ACC485614D13S305-FSTRPET-AAC TAA TGC AAG GAA ATT TGT GG355712300030015D13S305-RTGA GGA CCT GTC GTT ACG AAT485916D13S1817-FSTR6-FAM-ACC GGA CCT CAG ATC TGA AT505820500050017D13S1817-RTGC CTG AGT AAA GGA AGT GG505818D18S386-FSTRNED-TCA GGA GAA TCA CTT GGA AC455510250025019D18S386-RTCC ATG AAG TAG CTA AGC AG455520D18S390-FSTRVIC-GCC CAG GAT GAG GAG GTA AT55596150015021D18S390-RTCC TGG TCT GAG GTG TCA TT505922D18S391-FSTRVIC-TGG ACT TAC CAC AGG CAA TG50585125012523D18S391-RTTC ACT ATT CCC ATC TGA GTC AC445824D18S535-FSTRNED-AGA CAG AAA TAT AGA TGA GAA TGC A32578200020025D18S535-RTCA TGT GAC AAA AGC CAC AC455726D21S1411-FSTRVIC-ATG ATG AAT GCA TAG ATG GAT G365510250025027D21S1411-RAAT GTG TGT CCT TCC AGG C535828D21S1414-FSTR6-FAM-AAA TTA GTG TCT GGC ACC CAG TA446012300030029D21S1414-RCAA TTC CCC AAG TGA ATT GCC TTC466130D21S1435-FSTRPET-CCC TCT CAA TTG TTT GTC TAC C455713325032531D21S1435-RGCA AGA GAT TTC AGT GCC AT455732D21S1446-FSTRPET-ATG TAC GAT ACG TAA TAC TTG ACA A325712300030033D21S1446-RGTC CCA AAG GAC CTG CTC615834SRY-Ffs6-FAM-CGG AGA AGC TCT TCC TTC CT55596,51625162,535SRY-RTAA GTG GCC TAG CTG GTG CT556236MYPT2/Y-Ffs6-FAM-CTC ACT ACA TGA CAT TCA GG45545125012537MYPT2/Y-RGTT TCT TCC CAG TAT CTA GTA CAG TGC456238DYS448-FSTRPET-CAA GGA TCC AAA TAA AGA ACA GAG A365810250025039DYS448-RGGT TAT TTC TTG ATT CCC TGT G415640CDY1/2-FfsPET-TAT TGA GAC CCT TGC ACC TG505825005041CDY1/2-RGTT TCT TGG AGT TTC CCT TCT GTC ACC486542176,5μl+23.5μl H2O
ABCDEFGH1NameSTR or fixed size (fs)Sequence 5 `-&gt; 3`GC %Tm 0CAmount (in μl) used in the primer mix from 50 pmol/μl  of Forward/Reverse primer stocksConcentration in the QF-PCR mix (nM)Final concentration in the QF-PCR
  reaction (nM)2AMEL-X/Y-Ffs6-FAM-CCC TGG GCT CTG TAA AGA ATA GTG50612500503AMEL-X/Y-RATC AGA GCT TAA ACT GGG AAG CTG46624DXS6803-FSTRVIC-GAA ATG TGC TTT GAC AGG AA40551537503755DXS6803-RCAA AAA GGG ACA TAT GCT ACT T36556DXYS218-FSTRPET-AAT CTG GGC CTG AAG CAT TT45581025002507DXYS218-RAAG AGC GAA ACT CCG TCT CA50608TAF9B-Ffs6-FAM-TTT GAC AGG TAG TTT TGG GTC A41583750759TAF9B-R1TGG TTT TGC CTA GGT CCA GT506010XHPRT-FSTR6-FAM-ATG CCA CAG ATA ATA CAC ATC CCC46617175017511XHPRT-RCTC TCC AGA ATA GTT AGA TGT AGG425612D13S258-FSTRNED-ACC TGC CAA ATT TTA CCA GG45578200020013D13S258-RGAC AGA GAG AGG GAA TAA ACC485614D13S305-FSTRPET-AAC TAA TGC AAG GAA ATT TGT GG355712300030015D13S305-RTGA GGA CCT GTC GTT ACG AAT485916D13S1817-FSTR6-FAM-ACC GGA CCT CAG ATC TGA AT505820500050017D13S1817-RTGC CTG AGT AAA GGA AGT GG505818D18S386-FSTRNED-TCA GGA GAA TCA CTT GGA AC455510250025019D18S386-RTCC ATG AAG TAG CTA AGC AG455520D18S390-FSTRVIC-GCC CAG GAT GAG GAG GTA AT55596150015021D18S390-RTCC TGG TCT GAG GTG TCA TT505922D18S391-FSTRVIC-TGG ACT TAC CAC AGG CAA TG50585125012523D18S391-RTTC ACT ATT CCC ATC TGA GTC AC445824D18S535-FSTRNED-AGA CAG AAA TAT AGA TGA GAA TGC A32578200020025D18S535-RTCA TGT GAC AAA AGC CAC AC455726D21S1411-FSTRVIC-ATG ATG AAT GCA TAG ATG GAT G365510250025027D21S1411-RAAT GTG TGT CCT TCC AGG C535828D21S1414-FSTR6-FAM-AAA TTA GTG TCT GGC ACC CAG TA446012300030029D21S1414-RCAA TTC CCC AAG TGA ATT GCC TTC466130D21S1435-FSTRPET-CCC TCT CAA TTG TTT GTC TAC C455713325032531D21S1435-RGCA AGA GAT TTC AGT GCC AT455732D21S1446-FSTRPET-ATG TAC GAT ACG TAA TAC TTG ACA A325712300030033D21S1446-RGTC CCA AAG GAC CTG CTC615834SRY-Ffs6-FAM-CGG AGA AGC TCT TCC TTC CT55596,51625162,535SRY-RTAA GTG GCC TAG CTG GTG CT556236MYPT2/Y-Ffs6-FAM-CTC ACT ACA TGA CAT TCA GG45545125012537MYPT2/Y-RGTT TCT TCC CAG TAT CTA GTA CAG TGC456238DYS448-FSTRPET-CAA GGA TCC AAA TAA AGA ACA GAG A365810250025039DYS448-RGGT TAT TTC TTG ATT CCC TGT G415640CDY1/2-FfsPET-TAT TGA GAC CCT TGC ACC TG505825005041CDY1/2-RGTT TCT TGG AGT TTC CCT TCT GTC ACC486542176,5μl+23.5μl H2O
?. [Main QF-PCR reaction]
Perform main QF-PCR reaction in volume of 20 µl consisting of 1X AmpliTaq Gold 360 Buffer (Applied Biosystems Inc), 1.875 mM MgCl2, 187.5 µM each of the four dNTPs (dATP, dCTP, dGTP, and dTTP), 1.5 µl 360 GC Enhancer (Applied Biosystems Inc), 50-667 nM primers (Table 1), 0.75 U AmpliTaq Gold 360 DNA Polymerase (Applied Biosystems Inc) and 10-50 ng DNA (variable amount of DNA concentration with a minimum of 2 ng/µl for succsesful multiplex PCR). Mix for each sample:
H2O                                                                                       6,30 μl10х PCR Buffer                                                                    2 μl25 mM MgCl2                                                                      1,5 μl2.5mM dNTP                                                                       1,5 μlGC rich enchancer                                                           1,5 μlPCR primer mix                                                                    2 μlTaq Gold 360 DNA polymerase (5U⁄µL)                          0,2 μlDNA +  H2O(if if required)                                                   5 μlTotal                                                                                       20 μl Thermal cycling conditions:- Initial denaturation                               95oC  10'                               x 29 cycles - Denaturation                                          95oC  45"        - Annealing                                               58oC  1'   - Extension                                               72oC  1'30"- Final Extension                                     60 oC   30' - Final hold                                               4 oCPerform PCR reaction in Thermal Cycler ("AB 2720" or "Veriti 96 well"  Thermal Cyclers in our case).H2O                                                                                       6,30 μl10х PCR Buffer                                                                    2 μl25 mM MgCl2                                                                      1,5 μl2.5mM dNTP                                                                       1,5 μlGC rich enchancer                                                           1,5 μlPCR primer mix                                                                    2 μlTaq Gold 360 DNA polymerase (5U⁄µL)                          0,2 μlDNA +  H2O(if if required)                                                   5 μlTotal                                                                                       20 μl Thermal cycling conditions:- Initial denaturation                               95oC  10'                               x 29 cycles - Denaturation                                          95oC  45"        - Annealing                                               58oC  1'   - Extension                                               72oC  1'30"- Final Extension                                     60 oC   30' - Final hold                                               4 oCPerform PCR reaction in Thermal Cycler ("AB 2720" or "Veriti 96 well"  Thermal Cyclers in our case).- Initial denaturation                               95oC  10'                               x 29 cycles - Denaturation                                          95oC  45"        - Annealing                                               58oC  1'   - Extension                                               72oC  1'30"- Final Extension                                     60 oC   30' - Final hold                                               4 oC
?. [Main QF-PCR reaction]
Capillary electrophoresis of the PCR products is performed on ABI Genetic Analyzers, in our case with ABI 3500 and ABI 3130. The procedure for preparation of the sample for capillary electrophoresis is same for both instruments:Combine 1 µl of the finished PCR reaction  with 12 µl of Hi-Di Formamide and 0.15 µl of GeneScan 500 LIZ Size Standard;Denature on 95oC for  10'  and place on ice;Run on ABI Genetic analyzer for automatic capillary electrophoresis and analyze with GeneMapper analysis software according to manufacturer's protocol. Technical specifications for running the  automatic capillary electrophoresis on the ABI 3500 Genetic analyzer are:    - Polimer: POP-7;    - Capillary array: 50 cm, 8-Capillary;    - Dye Set: G5;    - Oven temperature: 60 oC;    - Injection voltage: 1.6 kVolts;    - PreRun voltage: 15 kvolts;    - PreRun time: 180 sec.;    - Injection time: 3 sec;    - Run voltage: 19.5 kvolts;    - Run time: 1200 sec.Technical specifications for running the  automatic capillary electrophoresis on the ABI 3130 Genetic analyzer are:    - Polimer: POP-4;    - Capillary array: 36 cm, 4-Capillary;    - Dye Set: G5;    - Oven temperature: 60 oC;    - Injection voltage: 1.2 kVolts;    - PreRun voltage: 15 kvolts;    - PreRun time: 180 sec.;    - Injection time: 8 sec;    - Run voltage: 15 kvolts;    - Run time: 2500 sec.Examples for electropherograms from normal male, normal female, Trisomy 21, Trisomy 18, Trisomy 13, Triploidy, Klinefelter sy., Turner sy. and Trisomy X samples are shown below:
?. [Additional QF-PCR reactions specific for given chromosome]
In this section, tecnical details are given about the chromosome specific backup markers. For chromosomes 13, 18 and 21, markers are combined in separate mixes, while for the sex chromosomes three markers are analyzed in single reactions.Steps for performing QF-PCR reactions specific for given chromosome are essentialy the same as  main QF-PCR reaction and consists of the following steps:3.1) Prepare QF-PCR multiplex primer mix / singleton mix;3.2) Perform PCR reaction;3.3) Capillary electrophoresis of the PCR product and analysis of the results.
?. [Additional QF-PCR reactions specific for given chromosome]
QF-PCR primer mixes for chromosomes 13, 18 and 21 are prepared in a total volume of 60 µl while primers for sex chromosomes are prepared as singletons with a concentration of 10 pmol/µl of the forward+reverse primer.Sequences of the primers, their GC percentages, melting points and amount of each primer used in preparation of the QF-PCR mix are shown in the folowing table:  ABCDEFGH1Target
  chromosomeNameSequence 5 `-&gt; 3`GC %Tm 0CAmount (in μl) used in the primer
  mix from 50 pmol/μl&nbsp; of Forward/Reverse
  primer stocksConcentration in the 
    QF-PCR mix (nM)Final concentration in the QF-PCR
  reaction 
    (nM)2Chr 13D13S742-FVIC-ATA ACT GGG CTA GGA ATG GAA ATA386043333.3166.73Chr 13D13S742-RGAC TTC CCA ATT CAG GAG GAC T5062.14Chr 13D13S628-F6-FAM-TAA CAT TCA TTG TCC CTT ACA GAT3358.31210000.0500.05Chr 13D13S628-RGCA AGG CTA TCT AAC GAT AAT TCA3860.36Chr 13D13S634-FVIC-TCC AGA TAG GCA GAT TCA AT4054.31613333.3666.77Chr 13D13S634-RCCT TCT TCT TCC CAT TGA TA4054.38Chr 13D13S631-FVIC-GGC AAC AAG AGC AAA ACT CT56.844543333.3166.79Chr 13D13S631-RTAG CCC TCA CCA TGA TTG G56.4752.6310Chr 13D13S258-FNED-ACC TGC CAA ATT TTA CCA GG4556.821666.783.311Chr 13D13S258-RGAC AGA GAG AGG GAA TAA ACC47.6255.712&nbsp;= 38 µl + 22 µl H2O13Chr 18D18S51-FNED-GAG CCA TGT TCA TGC CAC TG5560.543333.3166.714Chr 18D18S51-RCAA ACC CGA CTA CCA GCA AC5560.515Chr 18D18S1367-F6-FAM-TTG GTT CAT CCA AAC ATG GT4054.343333.3166.716Chr 18D18S1367-RATA ACT GCA GAG AAC GTT GC4556.417Chr 18D18S978-FVIC-TTC TTC AGT ATC ATC TTG TGC C4158.465000.0250.018Chr 18D18S978-RGCC AAATGT AGA TCT TGG GA4556.419Chr 18D18S391-FVIC-TGG ACT TAC CAC AGG CAA TG5058.421666.783.320Chr 18D18S391-RTTC ACT ATT CCC ATC TGA GTC AC43.558.221&nbsp;= 16 µl + 44 µl H2O22Chr 21D21S1437-FPET-ATG TAC ATG TGT CTG GGA AGG4859.543333.3166.723Chr 21D21S1437-RTTC TCT ACA TAT TTA CTG CCA ACA3358.324Chr 21D21S11-FNED-TAT GTG AGT CAA TTC CCC AAG TGA426243333.3166.725Chr 21D21S11-RGTT GTA TTA GTC AAT GTT CTC CAG3860.326Chr 21D21S1412-F6-FAM-CGG AGG TTG CAGTGA GTT G5859.543333.3166.727Chr 21D21S1412-RGGG AAGGCT ATG GAG GAG A5859.528Chr 21D21S1441-F6-FAM-CAA AGC TGC AGT GAG CTG TA5058.443333.3166.729Chr 21D21S1441-RCTC AGG GAA CTG ATG GTC AC5560.530Chr 21D21S1411-FVIC-ATG ATG AAT GCA TAG ATG GAT G36.454.843333.3166.731Chr 21D21S1411-RAAT GTG TGT CCT TCC AGG C52.658.232&nbsp;= 20 µl + 40 µl H2O33Chr X+YX-22-F6-FAM-TAA TGA GAG TTG GAA AGA AA3050.2//500.034Chr X+YX-22-RCCC ATT GTT GCT ACT TGA GA4556.435Chr XDXS6809-FVIC-TGA ACC TTC CTA GCT CAG GA5058.4//500.036Chr XDXS6809-RTCT GGA GAA TCC AAT TTT GC4054.337Chr XDXS996-FVIC -AAATTCTTGCTTAGGCCACTCTAGG4461//500.038Chr XDXS996-RGTTGTTCTGGATCGTATGCTAGG4859
ABCDEFGH1Target
  chromosomeNameSequence 5 `-&gt; 3`GC %Tm 0CAmount (in μl) used in the primer
  mix from 50 pmol/μl&nbsp; of Forward/Reverse
  primer stocksConcentration in the 
    QF-PCR mix (nM)Final concentration in the QF-PCR
  reaction 
    (nM)2Chr 13D13S742-FVIC-ATA ACT GGG CTA GGA ATG GAA ATA386043333.3166.73Chr 13D13S742-RGAC TTC CCA ATT CAG GAG GAC T5062.14Chr 13D13S628-F6-FAM-TAA CAT TCA TTG TCC CTT ACA GAT3358.31210000.0500.05Chr 13D13S628-RGCA AGG CTA TCT AAC GAT AAT TCA3860.36Chr 13D13S634-FVIC-TCC AGA TAG GCA GAT TCA AT4054.31613333.3666.77Chr 13D13S634-RCCT TCT TCT TCC CAT TGA TA4054.38Chr 13D13S631-FVIC-GGC AAC AAG AGC AAA ACT CT56.844543333.3166.79Chr 13D13S631-RTAG CCC TCA CCA TGA TTG G56.4752.6310Chr 13D13S258-FNED-ACC TGC CAA ATT TTA CCA GG4556.821666.783.311Chr 13D13S258-RGAC AGA GAG AGG GAA TAA ACC47.6255.712&nbsp;= 38 µl + 22 µl H2O13Chr 18D18S51-FNED-GAG CCA TGT TCA TGC CAC TG5560.543333.3166.714Chr 18D18S51-RCAA ACC CGA CTA CCA GCA AC5560.515Chr 18D18S1367-F6-FAM-TTG GTT CAT CCA AAC ATG GT4054.343333.3166.716Chr 18D18S1367-RATA ACT GCA GAG AAC GTT GC4556.417Chr 18D18S978-FVIC-TTC TTC AGT ATC ATC TTG TGC C4158.465000.0250.018Chr 18D18S978-RGCC AAATGT AGA TCT TGG GA4556.419Chr 18D18S391-FVIC-TGG ACT TAC CAC AGG CAA TG5058.421666.783.320Chr 18D18S391-RTTC ACT ATT CCC ATC TGA GTC AC43.558.221&nbsp;= 16 µl + 44 µl H2O22Chr 21D21S1437-FPET-ATG TAC ATG TGT CTG GGA AGG4859.543333.3166.723Chr 21D21S1437-RTTC TCT ACA TAT TTA CTG CCA ACA3358.324Chr 21D21S11-FNED-TAT GTG AGT CAA TTC CCC AAG TGA426243333.3166.725Chr 21D21S11-RGTT GTA TTA GTC AAT GTT CTC CAG3860.326Chr 21D21S1412-F6-FAM-CGG AGG TTG CAGTGA GTT G5859.543333.3166.727Chr 21D21S1412-RGGG AAGGCT ATG GAG GAG A5859.528Chr 21D21S1441-F6-FAM-CAA AGC TGC AGT GAG CTG TA5058.443333.3166.729Chr 21D21S1441-RCTC AGG GAA CTG ATG GTC AC5560.530Chr 21D21S1411-FVIC-ATG ATG AAT GCA TAG ATG GAT G36.454.843333.3166.731Chr 21D21S1411-RAAT GTG TGT CCT TCC AGG C52.658.232&nbsp;= 20 µl + 40 µl H2O33Chr X+YX-22-F6-FAM-TAA TGA GAG TTG GAA AGA AA3050.2//500.034Chr X+YX-22-RCCC ATT GTT GCT ACT TGA GA4556.435Chr XDXS6809-FVIC-TGA ACC TTC CTA GCT CAG GA5058.4//500.036Chr XDXS6809-RTCT GGA GAA TCC AAT TTT GC4054.337Chr XDXS996-FVIC -AAATTCTTGCTTAGGCCACTCTAGG4461//500.038Chr XDXS996-RGTTGTTCTGGATCGTATGCTAGG4859
?. [Additional QF-PCR reactions specific for given chromosome]
Perform main QF-PCR reaction in volume of 20 µl consisting of 1X AmpliTaq Gold 360 Buffer (Applied Biosystems Inc), 1.875 mM MgCl2, 187.5 µM each of the four dNTPs (dATP, dCTP, dGTP, and dTTP), 1.5 µl 360 GC Enhancer (Applied Biosystems Inc), 83-500 nM primers (Table 1), 0.75 U AmpliTaq Gold 360 DNA Polymerase (Applied Biosystems Inc) and 10-50 ng DNA (variable amount of DNA concentration with a minimum of 2 ng/µl for succsesful multiplex PCR). Mix for each sample:
H2O                                                                                       7,30 μl10х PCR Buffer                                                                    2 μl25 mM MgCl2                                                                      1,5 μl2.5mM dNTP                                                                       1,5 μlGC rich enchancer                                                              1,5 μlPCR primer mix                                                                    1 μlTaq Gold 360 DNA polymerase (5U⁄µL)                          0,2 μlDNA +  H2O(if required)                                                     5 μlTotal                                                                                       20 μl Thermal cycling conditions:- Initial denaturation                               95oC  10'                               x 29 cycles - Denaturation                                          95oC  45"        - Annealing                                               58oC  1'   - Extension                                               72oC  1'30"- Final Extension                                     60 oC   30' - Final hold                                               4 oCPerform PCR reaction in Thermal Cycler ("AB 2720" or "Veriti 96 well"  Thermal Cyclers in our case).H2O                                                                                       7,30 μl10х PCR Buffer                                                                    2 μl25 mM MgCl2                                                                      1,5 μl2.5mM dNTP                                                                       1,5 μlGC rich enchancer                                                              1,5 μlPCR primer mix                                                                    1 μlTaq Gold 360 DNA polymerase (5U⁄µL)                          0,2 μlDNA +  H2O(if required)                                                     5 μlTotal                                                                                       20 μl Thermal cycling conditions:- Initial denaturation                               95oC  10'                               x 29 cycles - Denaturation                                          95oC  45"        - Annealing                                               58oC  1'   - Extension                                               72oC  1'30"- Final Extension                                     60 oC   30' - Final hold                                               4 oCPerform PCR reaction in Thermal Cycler ("AB 2720" or "Veriti 96 well"  Thermal Cyclers in our case).- Initial denaturation                               95oC  10'                               x 29 cycles - Denaturation                                          95oC  45"        - Annealing                                               58oC  1'   - Extension                                               72oC  1'30"- Final Extension                                     60 oC   30' - Final hold                                               4 oC
?. [Additional QF-PCR reactions specific for given chromosome]
Capillary electrophoresis of the PCR products is performed on ABI Genetic Analyzers, in our case with ABI 3500 and ABI 3130. The procedure for preparation of the sample for capillary electrophoresis is same for both instruments:Combine 1 µl of the finished PCR reaction  with 12 µl of Hi-Di Formamide and 0.15 µl of GeneScan 500 LIZ Size Standard;Denature on 95oC for  10'  and place on ice;Run on ABI Genetic analyzer for automatic capillary electrophoresis and analyze with GeneMapper analysis software according to manufacturer's protocol. Technical specifications for running the automatic capillary electrophoresis on the ABI 3500 and ABI 3130 Genetic analyzers are same as for main QF-PCR reaction.Examples for electropherograms from samples with Trisomy 21, Trisomy 18 and Trisomy 13 are shown below: