Single PCR Multiplex SNaPshot Reaction for Detection of Eleven Blood Group Nucleotide Polymorphisms

Abstract

Hemagglutination-based assays have several clinical shortcomings. To overcome this difficulty, we have developed a multiplex-PCR SNaPshot assay adapted to the Southern French population, which includes individuals from sub-Saharan Africa and the Comoros archipelago. Single nucleotide polymorphisms (SNPs) associated with clinically relevant blood antigens as well as with null phenotypes were profiled (i.e., K/k, Fy(a)/Fy(b)/Fy(bw)/Fy(null), S/s/U-/U+(var), Jk(a)/Jk(b), Do(a)/Do(b), Yt(a)/Yt(b), and Co(a)/Co(b)). A single multiplex-PCR reaction was used to amplify nine gene regions encompassing 11 SNPs. Identification was obtained by incorporation of the complementary dye-conjugated single base at the 3' end of each probe primer annealed proximal to the target SNP. After optimization, the SNaPshot assay was validated with 265 known allele or phenotype pairs. Results were found fully concordant with those of hemagglutination, allele-specific PCR, and/or sequencing. The assay was then evaluated on 227 blood samples in a clinical context. A total of 203 derived-phenotypes were generated, including 82 atypical phenotypes [i.e., Fy(b+(w)) (n = 32); K(+) (n = 22); Co(b+) (n = 8); Yt(b+) (n = 18); S-s+U+(var) (n = 2), 105 null phenotypes, i.e., Fy(a-b-) (n = 97); S-s-U- (n = 6); S-s-U+(var) (n = 2)] and sixteen Fy-positive samples carried a FY*Fy allele. The findings show that this assay can provide a low-cost and fast genotyping tool well adapted to local ethnically mixed populations.

Serum and red-blood-cell folate demonstrate differential associations with BMI in pregnant women

“…Samples were centrifuged at 1100g and DNA was extracted using the BioRobot M48 and MagAttract DNA Blood Midi Kit (Qiagen, Hilden, Germany). PCR was used to amplify DNA regions of interest and genotyping was performed using the ABI 3130xl Genetic Analyzer and the ABI Prism SnaPshot Multiplex Kit (Applied Biosystems Inc., Foster City, CA, USA), which allows genotyping of several SNP in the same reaction (12) . The laboratory procedures for genotyping have been described previously (11,13) .…”

Association between microRNA polymorphisms and humoral immunity to hepatitis B vaccine

“…Genotyping of these SNPs was performed by multiplex SNaPshot technology as previously described, 26,27 using an ABI fluorescence-based assay discrimination method (Applied Biosystems). The multiplex SNaPshot detection of single-base extended probe primers was based on fluorescence and extended length detected by capillary electrophoresis on an ABI3130XL Sequencer (Applied Biosystems).…”

Clinical Potential of Effective Noninvasive Exclusion of <b><i>KEL1</i></b>-Positive Fetuses in <b><i>KEL1</i></b>-Negative Pregnant Women

“…Following purification of the PCR products, enzymatic purification using exonuclease (Exonuclease I; Thermo Scientific, USA) and alkaline phosphatase (FastAP Thermosensitive Alkaline Phosphatase; Thermo Scientific, USA) was performed according to the manufacturer's instructions. A minisequencing extension reaction was performed using the SBE primer: 24T-TGG TAA ATG GAC TTC CTT AAA CTT TAA CCG AA [10]. Amplification was performed using the ABI PRISM SNaPshot Multiplex Kit (Applied Biosystems, USA) in a final volume of 8 μl.…”

“…The single nucleotide change 578C>T results in the substitution of threonine (‘k') to methionine (‘K') at position 193 (p.Thr193Met) [6,7]. For the detection of particular alleles, allele-specific polymerase chain reaction (AS-PCR) or PCR with subsequent cleavage by restriction enzyme Bsm I (PCR-RFLP) may be used [5,8,9,10,11]. It is also possible to use more sensitive and faster detection methods, such as allelic discrimination using TaqMan PCR, minisequencing using the SNaPshot system (Applied Biosystems, USA) or a DNA microarray platform [7,10].…”

“…For the detection of particular alleles, allele-specific polymerase chain reaction (AS-PCR) or PCR with subsequent cleavage by restriction enzyme Bsm I (PCR-RFLP) may be used [5,8,9,10,11]. It is also possible to use more sensitive and faster detection methods, such as allelic discrimination using TaqMan PCR, minisequencing using the SNaPshot system (Applied Biosystems, USA) or a DNA microarray platform [7,10]. Briefly, minisequencing is composed of PCR amplification for the formation of PCR products containing the target SNP and a single base extension (SBE) of the oligonucleotide primer using a fluorescently labeled dideoxynucleotide base at the 3′ end of each primer located proximal to the target SNP (minisequencing PCR).…”