Multiplex Preamplification Research Articles

IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

Enrichment of Mutations in Multiple DNA Sequences Using COLD-PCR in Emulsion

BackgroundMultiplex detection of low-level mutant alleles in the presence of wild-type DNA would be useful for several fields of medicine including cancer, pre-natal diagnosis and infectious diseases. COLD-PCR is a recently developed method that enriches low-level mutations during PCR cycling, thus enhancing downstream detection without the need for special reagents or equipment. The approach relies on the differential denaturation of DNA strands which contain Tm-lowering mutations or mismatches, versus ‘homo-duplex’ wild-type DNA. Enabling multiplex-COLD-PCR that can enrich mutations in several amplicons simultaneously is desirable but technically difficult to accomplish. Here we describe the proof of principle of an emulsion-PCR based approach that demonstrates the feasibility of multiplexed-COLD-PCR within a single tube, using commercially available mutated cell lines. This method works best with short amplicons; therefore, it could potentially be used on highly fragmented samples obtained from biological material or FFPE specimens.MethodsFollowing a multiplex pre-amplification of TP53 exons from genomic DNA, emulsions which incorporate the multiplex product, PCR reagents and primers specific for a given TP53 exon are prepared. Emulsions with different TP53 targets are then combined in a single tube and a fast-COLD-PCR program that gradually ramps up the denaturation temperature over several PCR cycles is applied (temperature-tolerant, TT-fast-eCOLD-PCR). The range of denaturation temperatures applied encompasses the critical denaturation temperature (Tc) corresponding to all the amplicons included in the reaction, resulting to a gradual enrichment of mutations within all amplicons encompassed by emulsion.ResultsValidation for TT-fast-eCOLD-PCR is provided for TP53 exons 6–9. Using dilutions of mutated cell-line into wild-type DNA, we demonstrate simultaneous mutation enrichment between 7 to 15-fold in all amplicons examined.ConclusionsTT-fast-eCOLD-PCR expands the versatility of COLD-PCR and enables high-throughput enrichment of low-level mutant alleles over multiple sequences in a single tube.

Erratum to: Improving non-invasive genotyping in capercaillie (Tetrao urogallus): redesigning sexing and microsatellite primers to increase efficiency on faeces samples

A set of 14 microsatellite loci were used for amplification of faecal DNA from capercaillie (Tetrao urogallus). New internal primers were designed for each locus and were employed to perform a seminested PCR approach combined with a multiplex preamplification method. The aim of this process was to increase the quality of the multilocus genotypes and to reduce the amount of sample required. Values of allelic dropout and false alleles found after three repetitions were 8.1 and 1.4% respectively, and 52.63% of the samples amplified for a minimum of 12 loci. Additionally, capercaillie specific sex primers have been designed. These primers give short products suitable for degraded faecal sample amplification. New primers resulted in 87.5% of samples being successfully sexed, a value seven times higher than using the original P2/P8 CHD sexing method.

Abstract LB-410: Targeted resequencing of genes frequently mutated in esophageal adenocarcinoma using highly multiplexed Ion Torrent AmpliSeqTM technology and semiconductor sequencing

Abstract Introduction: Whole genome and whole exome sequencing by large collaborative research groups is providing us with an unprecedented view of the mutations that occur in cancer. Identifying clinically-relevant and actionable mutations however remains a challenge that will most likely be met by individual groups focused on specific clinical questions and scenarios. Such studies will rely on targeted resequencing of selected genes in large numbers of well-annotated clinical specimens. These specimens are most often formalin-fixed and paraffin-embedded (FFPE) and some of the most valuable samples are small biopsies or fine needle aspirates (FNA's). Translational and clinical researchers require rapid and flexible resequencing approaches that are compatible with the small amounts of highly degraded nucleic acids derived from these challenging samples. Materials & Methods: Using Illumina whole exome sequence data from >60 esophageal adenocarcinoma (EAC) specimens, we identified a set of 20 genes that are frequently mutated in this tumor. PCR amplicons no larger than 140bp were designed to cover all exons from these genes, using the Ion Torrent AmpliSeqTM Designer pipeline. Primers were pooled for a 1285-plex multiplex PCR preamplification step starting with 1-20ng of input DNA from both FFPE and frozen tissue and the resulting amplicon products were used to generate sequencing libraries. Semiconductor sequencing was performed on the Ion Personal Genome MachineTM (PGMTM) sequencer. Metrics evaluated included reads on target, base accuracy, uniform coverage of bases and sequencing base read depth. Mutations detected in EAC samples were compared between the two sequencing platforms. Data from dysplastic and metaplastic esophageal biopsies are now being generated to identify mutations that occur in the development of EAC. Results: 1285 PCR primer pairs were designed that covered 66103bp of the coding region of the 20 target genes. Sequencing data covered >99% with at least 100 reads indicating minimal drop-out during preamplification. Average base read depth was >1900X and no significant differences were observed between FFPE and frozen tissue derived DNA. Initial studies demonstrate 100% concordance of mutations (n=42) detected in the 20 target genes and mutant allele frequencies are highly correlated between the two sequencing approaches (slope =0.96; R2 =0.85; range 4-78%). Conclusion: We have used the Ion Torrent AmpliSeqTM technology to detect mutations in EAC specimens and precursor lesions with high sensitivity and accuracy. The flexible primer design pipeline combined with AmpliSeqTM technology provides a fast and easy approach to targeted resequencing in particularly challenging clinical specimens including FFPE biopsies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-410. doi:1538-7445.AM2012-LB-410

DNA Extracted from Stained Sputum Smears Can Be Used in the MTBDR plus Assay

We examined the feasibility of using DNA extracted from stained sputum smears for the detection of rifampin and isoniazid resistance with the commercial MTBDRplus assay from Hain Lifescience GmbH, Nehren, Germany. Overall sensitivity was initially low (70.0%) but increased to 96.7% when a multiplex PCR preamplification step was added. We then tested stored Mycobacterium tuberculosis-positive stained smears prepared from 297 patients' sputum samples. Species identification and drug susceptibility testing (DST) had been performed at the Institut Pasteur de Madagascar. Overall, the performance of the MTBDRplus assay applied to slide DNA was similar to that obtained in other studies with DNA extracted from clinical specimens. With the ready availability of stained smears in routine diagnostic laboratories and their easy transport and storage at room temperature, this approach should be useful for optimizing the treatment of multidrug-resistant tuberculosis and for conducting resistance surveys aimed at identifying hot-spot regions and breaking chains of transmission.

Multiplex preamplification PCR and microsatellite validation enables accurate single nucleotide polymorphism genotyping of historical fish scales

Incorporating historical tissues into the study of ecological, conservation and management questions can broaden the scope of population genetic research by enhancing our understanding of evolutionary processes and anthropogenic influences on natural populations. Genotyping historical and low-quality samples has been plagued by challenges associated with low amounts of template DNA and the potential for pre-existing DNA contamination among samples. We describe a two-step process designed to (i) accurately genotype large numbers of historical low-quality scale samples in a high-throughput format and (ii) screen samples for pre-existing DNA contamination. First, we describe how an efficient multiplex preamplification PCR of 45 single nucleotide polymorphisms (SNPs) can generate highly accurate genotypes with low failure and error rates in subsequent SNP genotyping reactions of individual historical scales from sockeye salmon (Oncorhynchus nerka). Second, we demonstrate how the method can be modified for the amplification of microsatellite loci to detect pre-existing DNA contamination. A total of 760 individual historical scale and 182 contemporary fin clip samples were genotyped and screened for contamination. Genotyping failure and error rates were exceedingly low and similar for both historical and contemporary samples. Pre-existing contamination in 21% of the historical samples was successfully identified by screening the amplified microsatellite loci. The advantages of automation, low failure and error rates, and ability to multiplex both the preamplification and subsequent genotyping reactions combine to make the protocol ideally suited for efficiently genotyping large numbers of potentially contaminated low-quality sources of DNA.

Multiplex pre‐amplification for noninvasive genetic sampling: is the extra effort worth it?

Microsatellite genotyping of hair and faeces using standard polymerase chain reaction (PCR) resulted in low success rates and high error rates in a 2003-2004 pilot study using noninvasive genetic sampling for the brown bear (Ursus arctos) in the Italian Alps. Thus, we evaluated the performance of multiplex pre-amplification for improving microsatellite genotyping results. Brown bear faecal DNA extracts of varying quality (n = 33) and hair DNA extracts of poor (n = 32) and good (n = 34) quality were used to compare standard PCR and pre-amplification. In contrast to previous studies, there was no significant difference between methods for individual locus amplification success, genotyping error and genotyping success rates for scat and hair samples. The use of pre-amplification requires an additional investment of time and resources, and our results raise questions about the universal value of pre-amplification approaches. We suggest that researchers carefully evaluate the performance of pre-amplification compared to standard PCR using field-collected samples from the study area of interest before engaging in large-scale noninvasive genetic analyses.

Abstract 1182: Single cell analysis of primary and disseminated tumor cells isolated from tissue and blood

Abstract Background: Cancer evolves through the accumulation of multiple genetic alterations. How such alterations are related to tumor growth and metastatic dissemination is being widely investigated. Tissue level analyses, however, may be confounded by tumor cell heterogeneity and contamination by wildtype stromal and immune cells. Single cell analysis of tumor cells may contribute to a better understanding of tumorigenesis and metastasis. Single cell analyses using flow cytometry or microdissected cells from tissues require large numbers of cells, expensive equipment, laborious technical training, and the analyzed single cell information may be impacted by technical processing artifacts. We developed an efficient, reliable and simple new method to isolate fresh single tumor cells from tissues and blood for mutational analysis and to evaluate additional biomarkers. Materials and Methods: Tumor tissues and blood samples were obtained from breast cancer patients after informed consent. Primary or metastatic tissue was used for preparation of single cell suspensions for subsequent isolation. Single cells were isolated using EpCAM-conjugated microbeads and the MagSweeper, a device invented by our group. Individual microbead-labeled single cells were easy to observe and extract. Single cells were placed into individual PCR tubes for further DNA and RNA analyses. Single cell DNA mutational analysis was performed by sequencing using multiplex pre-amplification followed by PCR amplification. Multiplex RNA expression analyses were performed by qRT-PCR from single tumor cells. Enriched and unenriched single cell suspensions were used for biomarker evaluation and results were compared with normal tissues. Tumor cells with mutations were compared on a single cell level among multiple compartments: primary tumor, blood, and metastatic tumor. Results and Discussion: Single cells were isolated from suspensions prepared from primary and metastatic tumor tissues, EpCAM-enriched and unenriched single cells microscopically observed without requiring additional treatment and used for immunostaining for tumor cells identification. Some single cells not bound to EpCAM microbeads still met tumor cell criteria (cytokeratin positive, CD45 negative, and DAPI positive). The percentage of EpCAM -positive and cytokeratin-positive was determined in the single cell suspensions. Single cell DNA showing a single nucleotide mutation in the PIK3CA gene was identified in individual tumor cells from different tissues but not white blood cells. Results from our model indicate that isolating single tumor cells from tumor tissues or blood provides more detailed information about different tumor compartments in individual cancer patients. Comparison of individual tumor cells from primary and/or metastatic sites to tumor cells in blood should provide further insight into the metastatic process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1182.

Highly efficient multiplex PCR of noninvasive DNA does not require pre‐amplification

Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.

Analysis of DNA Methylation of Multiple Genes in Microdissected Cells From Formalin-fixed and Paraffin-embedded Tissues

A procedure for simultaneous quantification of DNA methylation of several genes in minute amounts of sample material was developed and applied to microdissected formalin-fixed and paraffin-embedded breast tissues. The procedure is comprised of an optimized bisulfite treatment protocol suitable for samples containing only few cells, a multiplex preamplification and subsequent locus specific reamplification, and a novel quantitative bisulfite sequencing method based on the incorporation of a normalization domain into the PCR product. A real-time PCR assay amplifying repetitive elements was established to quantify low amounts of bisulfite-treated DNA. Ten prognostic and diagnostic epigenetic breast cancer biomarkers (PITX2, RASSF1A, PLAU, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2, and TFF1) were analyzed in tissue samples obtained from two patients with invasive ductal carcinoma of the breast. The microdissected samples were obtained from several areas within the tumor tissue, including intraductal and invasive carcinoma, adenosis, and normal ductal epithelia of adjacent normal tissue, as well as stroma, tumor infiltrating lymphocytes, and adipose tissue. Overall, reliable quantification was possible for all genes. For most genes, increased DNA methylation in invasive and intraductal carcinoma cells compared with other tissue components was observed. For TFF1, decreased methylation levels were observed in tumor cells.

Exon-Level Expression Profiling: A Comprehensive Transcriptome Analysis of Oral Fluids

The application of global gene expression profiling to saliva samples is hampered by the presence of partially fragmented and degraded RNAs that are difficult to amplify and detect with the prevailing technologies. Moreover, the often limited volume of saliva samples is a challenge to quantitative PCR (qPCR) validation of multiple candidates. The aim of this study was to provide proof-of-concept data on the combination of a universal mRNA-amplification method with exon arrays for candidate selection and a multiplex preamplification method for easy validation. We used a universal mRNA-specific linear-amplification strategy in combination with Affymetrix Exon Arrays to amplify salivary RNA from 18 healthy individuals on the nanogram scale. Multiple selected candidates were preamplified in one multiplex reverse transcription PCR reaction, cleaned up enzymatically, and validated by qPCR. We defined a salivary exon core transcriptome (SECT) containing 851 transcripts of genes that have highly similar expression profiles in healthy individuals. A subset of the SECT transcripts was verified by qPCR analysis. Informatics analysis of the SECT revealed several functional clusters and sequence motifs. Sex-specific salivary exon biomarkers were identified and validated in tests with samples from healthy individuals. It is feasible to use samples containing fragmented RNAs to conduct high-resolution expression profiling with coverage of the entire transcriptome and to validate multiple targets from limited amounts of sample.

Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

BackgroundDue to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan® PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp).ResultsT-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ± 0.33 Ct values. The coefficients for inter- (1.89 ± 0.48%) and intra-assay variation (0.85 ± 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 ± 0.33, without significant differences between self-designed and ABI inventoried Taqman® gene assays. Only two of the tested Taqman® ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 ± 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ± 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ± 0.74), albeit higher inter-assay CVs (5.38 ± 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.ConclusionIn comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

Multiplex preamplification of specific cDNA targets prior to gene expression analysis by TaqMan Arrays

BackgroundAn accurate gene expression quantification using TaqMan Arrays (TA) could be limited by the low RNA quantity obtained from some clinical samples. The novel cDNA preamplification system, the TaqMan PreAmp Master Mix kit (TPAMMK), enables a multiplex preamplification of cDNA targets and therefore, could provide a sufficient amount of specific amplicons for their posterior analysis on TA.FindingsA multiplex preamplification of 47 genes was performed in 22 samples prior to their analysis by TA, and relative gene expression levels of non-preamplified (NPA) and preamplified (PA) samples were compared. Overall, the mean cycle threshold (CT) decrement in the PA genes was 3.85 (ranging from 2.07 to 5.01). A high correlation (r) between the gene expression measurements of NPA and PA samples was found (mean r = 0.970, ranging from 0.937 to 0.994; p < 0.001 in all selected cases). High correlation coefficients between NPA and PA samples were also obtained in the analysis of genes from degraded RNA samples and/or low abundance expressed genes.ConclusionWe demonstrate that cDNA preamplification using the TPAMMK before TA analysis is a reliable approach to simultaneously measure gene expression of multiple targets in a single sample. Moreover, this procedure was validated in genes from degraded RNA samples and low abundance expressed genes. This combined methodology could have wide applications in clinical research, where scarce amounts of degraded RNA are usually obtained and several genes need to be quantified in each sample.

A molecular expression signature distinguishing follicular lesions in thyroid carcinoma using preamplification RT-PCR in archival samples

Follicular variant of papillary thyroid carcinoma is a lesion that frequently causes difficulties from a diagnostic perspective in the laboratory. The purpose of this study was to interrogate a cohort of archival thyroid lesions using gene expression analysis of a panel of markers proposed to have utility as adjunctive markers in the diagnosis of thyroid neoplasia and follicular variant of papillary thyroid carcinoma in particular. Laser Capture Microdissection was used to procure pure cell populations for extraction. In addition a novel, multiplex preamplification technique was used to facilitate analysis of multiple targets. The panel comprised: HLA-DMA, HLA-DBQ1, CD74, CSNK1G2, IRF3, KRAS2, LYN, MT1K, MT1X, RAB23, TGFB1 and TOP2A, with CDKN1B as an endogenous control. Expression profiles for each target were generated using TaqMan® Real-Time PCR. HLA-DMA, HLA-DQB1, MT1X, CSNK1G2 and RAB23 were found to be differentially expressed (P<0.05) when comparing follicular adenoma and follicular variant of papillary thyroid carcinoma. Comparison of follicular adenoma and follicular thyroid carcinoma groups showed significant differential expression for MT1K, MT1X and RAB23 (P<0.05). Comparison of the papillary thyroid carcinoma group (classic and follicular variants) and the follicular adenoma group showed differential expression for CSNK1G2, HLA-DQB1, MT1X and RAB23 (P<0.05). Finally, KRAS2 was found to be differentially expressed (P<0.05) when comparing the papillary thyroid carcinoma and follicular thyroid carcinoma groups. This panel of molecular targets discriminates between follicular adenoma, papillary thyroid carcinoma, follicular variant of papillary thyroid carcinoma and follicular thyroid carcinoma by their expression repertoires. It may have utility for broader use in the setting of fine-needle aspiration cytology and could improve the definitive diagnosis of certain categories of thyroid malignancy.

A test of the multiplex pre-amplification approach in microsatellite genotyping of wolverine faecal DNA

Recently, a two-step PCR approach, referred to as multiplex pre-amplification, was proposed to improve microsatellite amplification from non-invasive samples such as faecal DNA. Here, we compare this new approach to standard PCR with respect to amplification success and genotyping error rates in microsatellite analysis (18 markers) of wolverine faecal DNA (48 extracts initially shown to contain amplifiable DNA). The multiplex pre-amplification approach was clearly advantageous both in terms of successful PCR amplifications (91% vs. 80%) and allelic dropout rate (2.4% vs. 12.5%). However, dropouts were to a high extent repeated in all second-step amplifications following multiplex pre-amplification, indicative of being generated during the initial PCR. Analysing more than one PCR from the initial multiplex PCR product may thus be of limited value. We instead suggest to perform two initial multiplex PCRs and to analyse a single second-step PCR from each of them. This was tested for 22 extracts at 18 loci and proved to be an effective way to obtaining a correct genotype.

Improved noninvasive genotyping method: application to brown bear (Ursus arctos) faeces

AbstractWe redesigned new microsatellite primers and one sex‐specific primer for amplification of faecal DNA from brown bears (Ursus arctos). We also combined a semi‐nested polymerase chain reaction (PCR) with a newly developed multiplex preamplification method in order to increase the quality of the amplified DNA fragments. In comparison with a conventional PCR approach, the genotyping error rate was substantially reduced and the amplification rate was increased. This new approach could be transposed to other species where conventional PCR methods experience low success due to limited DNA concentration and/or quality.

A Multiplex Pre-Amplification Method that Significantly Improves Microsatellite Amplification and Error Rates for Faecal DNA in Limiting Conditions

A Multiplex Pre-Amplification Method that Significantly Improves Microsatellite Amplification and Error Rates for Faecal DNA in Limiting Conditions

Sex determination of bovine embryo blastomeres by fluorogenic probes

One of the major challenges of using genetic information in marker assisted selection (MAS) is the detection of multiple marker loci from a small biopsy sample of a preimplantation stage embryo. The objective of this study was to develop a fast, nested, multiplex preamplification, polymerase chain reaction (PCR) method for the determination of sex in bovine embryo blastomeres. For this aim, ZFX/ZFY sequences were preamplified simultaneously with other genomic regions. The preamplification product was used as a template in an allelic discrimination assay, with nested primers and sex specific fluorogenic probes for ZFX and ZFY. Fluorogenic probes were used to eliminate the need for time consuming electrophoresis. Compared to sexing with Bovy/κ-casein co-amplification method and other replicates from the same embryo, the accuracy of sexing with the use of fluorogenic probes after preamplification was 99% (112/113 blastomeres). The amplification efficiency was 96% (113/117 blastomeres).