Short PCR Amplicons Research Articles

Verification of CRISPR editing and finding transgenic inserts by Xdrop indirect sequence capture followed by short- and long-read sequencing.

Validation of CRISPR-Cas9 editing typically explores the immediate vicinity of the gene editing site and distal off-target sequences, which has led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop™, a new microfluidic technology that allows targeted enrichment of long regions (~100kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene-edited region in four cell lines on long- and short-read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kilobases-large insertions in three of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.

MHC genotyping from rhesus macaque exome sequences.

Indian rhesus macaque major histocompatibility complex (MHC) variation can influence the outcomes of transplantation and infectious disease studies. Frequently, rhesus macaques are MHC genotyped to identify variants that could account for unexpected results. Since the MHC is only one region in the genome where variation could impact experimental outcomes, strategies for simultaneously profiling variation in the macaque MHC and the remainder of the protein coding genome would be useful. Here we determine MHC class I and class II genotypes using target-capture probes enriched for MHC sequences, a method we term macaque exome sequence (MES) genotyping. For a cohort of 27 Indian rhesus macaques, we describe two methods for obtaining MHC genotypes from MES data and demonstrate that the MHC class I and class II genotyping results obtained with these methods are 98.1% and 98.7% concordant, respectively, with expected MHC genotypes. In contrast, conventional MHC genotyping results obtained by deep sequencing of short multiplex PCR amplicons were only 92.6% concordant with expectations for this cohort.

Hindering the illegal trade in dog and cat furs through a DNA-based protocol for species identification.

In Western countries dogs and cats are the most popular pets, and people are increasingly opposed to their rearing for the fur industry. In 2007, a Regulation of the European Union (EU) banned the use and trade of dog and cat furs, but an official analytical protocol to identify them as source species was not provided, and violations of law are still frequent in all Member States. In this paper we report on the development and validation of a simple and affordable DNA method for species detection in furs to use as an effective tool to combat illegal trade in fur products. A set of mitochondrial primers was designed for amplification of partial cytochrome b, control region and ND1 gene in highly degraded samples, like furs and pelts. Our amplification workflow involved the use of a non-specific primer pair to perform a first test to identify the species through sequencing, then the application of species-specific primer pairs to use in singleplex end-point PCRs as confirmation tests. The advantage of this two-step procedure is twofold: on the one hand it minimises the possibility of negative test results from degraded samples, since failure of amplification with a first set of primers can be offset by successful amplification of the second, and on the other it adds confidence and reliability to final authentication of species. All designed primers were validated on a reference collection of tissue samples, obtaining solid results in terms of specificity, sensitivity, repeatability and reproducibility. Application of the protocol on real caseworks from seized furs yielded successful results also from old and dyed furs, suggesting that age and chemical staining do not necessarily affect positive amplifications. Major pros of this approach are: (1) sensitive and informative primer sets for detection of species; (2) short PCR amplicons for the analysis of poor quality DNA; (3) binding primers that avoid contamination from human DNA; (4) user-friendly protocol for any laboratory equipped for analysis of low-copy-number DNA. Our molecular procedure proved to be a good starting point for enforcing the EU Regulation against dog and cat fur trade in forensic contexts where source attribution is essential to the assignment of responsibilities.

Homology-based enzymatic DNA fragment assembly-based illumina sequencing library preparation.

The current Illumina HiSeq and MiSeq platforms can generate paired-end reads of up to 2 x 250 bp and 2 x 300 bp in length, respectively. These read lengths may be substantially longer than genomic regions of interest when a DNA sequencing library is prepared through a target enrichment-based approach. A sequencing library preparation method has been developed based on the homology-based enzymatic DNA fragment assembly scheme to allow processing of multiple PCR products within a single read. Target sequences were amplified using locus-specific PCR primers with 8 bp tags, and using the tags, homology-based enzymatic DNA assembly was performed with DNA polymerase, T7 exonuclease and T4 DNA ligase. Short PCR amplicons can hence be assembled into a single molecule, along with sequencing adapters specific to the Illumina platforms. As a proof-of-concept experiment, short PCR amplicons (57–66 bp in length) derived from genomic DNA templates of field pea and containing variable nucleotide locations were assembled and sequenced on the MiSeq platform. The results were validated with other genotyping methods. When 5 PCR amplicons were assembled, 4.3 targeted sequences (single-nucleotide polymorphisms) on average were successfully identified within each read. The utility of this for sequencing of short fragments has consequently been demonstrated.

Improved sensitivity of circulating tumor DNA measurement using short PCR amplicons

Circulating tumor DNA is being extensively investigated as a clinically relevant cancer marker. KRAS mutations are present in 40% of colorectal tumors and monitoring the mutational status together with the level of mutated DNA is of great interest. The measurement of DNA from plasma or serum, however, presents a number of challenges that require attention. The amount of DNA is low and highly fragmented and analyses need to be optimized accordingly.KRAS ARMS-qPCR assays with amplicon lengths of 120 and 85 base pairs, respectively, were compared using positive control material (PCR fragments) and plasma samples from 46 colorectal cancer patients known to harbor a tumor KRAS mutation.KRAS mutated DNA was detected in significantly more clinical samples using the short amplicon assays compared to the long amplicon assays (74% vs. 61%, p=0.03). The level of mutated DNA in plasma was on average three times higher using short amplicon assays.Our results reflect the importance of minimizing the assay length when analyzing highly fragmented DNA, especially if these analyses are to be used for treatment monitoring and relapse detection.

Detection and Identification of Salmonella enterica, Escherichia coli, and Shigella spp. via PCR-Electrospray Ionization Mass Spectrometry: Isolate Testing and Analysis of Food Samples

An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay's ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens.

Polyadenylated Sequencing Primers Enable Complete Readability of PCR Amplicons Analyzed by Dideoxynucleotide Sequencing

Dideoxynucleotide DNA sequencing is one of the principal procedures in molecular biology. Loss of an initial part of nucleotides behind the 3' end of the sequencing primer limits the readability of sequenced amplicons. We present a method which extends the readability by using sequencing primers modified by polyadenylated tails attached to their 5' ends. Performing a polymerase chain reaction, we amplified eight amplicons of six human genes (AMELX, APOE, HFE, MBL2, SERPINA1 and TGFB1) ranging from 106 bp to 680 bp. Polyadenylation of the sequencing primers minimized the loss of bases in all amplicons. Complete sequences of shorter products (AMELX 106 bp, SERPINA1 121 bp, HFE 208 bp, APOE 244 bp, MBL2 317 bp) were obtained. In addition, in the case of TGFB1 products (366 bp, 432 bp, and 680 bp, respectively), the lengths of sequencing readings were significantly longer if adenylated primers were used. Thus, single strand dideoxynucleotide sequencing with adenylated primers enables complete or near complete readability of short PCR amplicons.

Detection of banned ruminant-derived material in industrial feedstuffs by TaqMan real-time PCR Assay.

A ruminant-specific real-time PCR system was designed and applied for the detection of processed animal protein from ruminants in industrial feedstuffs. The assay includes a primer pair and a TaqMan probe selectively targeting mitochondrial 16S rRNA gene sequences from the ruminant group and another primer-probe set based on the eukaryotic nuclear 18S rRNA gene (positive amplification control). Both ruminant and eukaryotic PCR systems generated short PCR amplicons of 79 and 77 bp, respectively. To evaluate the suitability of the real-time PCR assay for the detection of banned by-products of ruminant origin, 126 feed samples subjected to rendering under current European legislation regulations were analyzed. The assay achieved 100% success in classifying the samples as positive or negative in terms of qualitative ruminant composition, with a detection limit of 0.1%. The quantitative ability of the assay is however restricted by variations in the composition and treatment of the feeds, which affect the amount and quality of amplifiable DNA.

HRM technology for the identification and characterization of INDEL and SNPs mutations in genes involved in drought and salt tolerance of durum wheat

WRKY transcription factors are one of the largest families of transcriptional regulators and form an integral part of signalling webs which modulate many plant processes, such as abiotic stress tolerance. In the present paper, an innovative method has been applied to identify novel WRKY-1 alleles involved in the responses to salt and drought stresses in Triticum durum. This technique involves scanning for sequencing variations in cDNA-derived PCR amplicons, using high-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM represents a novel advance in detection of single-nucleotide polymorphisms (SNPs) by measuring temperature-induced strand separation of short PCR amplicons. The use of this approach is still limited in the field of plant biology. Here, HRM analysis has been applied to the discovery and genotyping of durum wheat SNPs. Specific primers have been designed, starting at multi-alignment of WRKY-1-conserved portions. The PCR amplicons, containing single SNPs, produce distinctive HRM profiles, and by sequencing the PCR products identified, SNPs have been characterized and validated. The results showed that all the revealed SNPs are located on salt-tolerant varieties, confirming their value in breeding activities.

High Resolution Melting Analysis for Rapid JAK2 Exon 14 and Exon12 Mutation Detection In Formalin Fixed Paraffin-Embeded Trephine Bone Marrow Biopsies of Patients with Polycythemia Vera. Detection of Novel JAK2 mutations

AbstractAbstract 1984Polycythemia vera (PV) belongs to the group of Ph1 negative myeloproliferative neoplasms and is characterized by the presence of JAK2V617F somatic mutation in >95% of PV patients. In some patients lacking this mutation, alternative mutations in exon 12 of the JAK2 gene have been reported. Identification of JAK2 mutations is a major WHO criterion for diagnosis of PV. Therefore, reliable and sensitive methods are needed for detection of these mutations. High resolution melting analysis (HRM) for mutation detection in JAK2 exons 12 and 14 have been previously described using DNA isolated from fresh patient samples. We report here the development of an HRM screening method for both JAK2 exons 12 and 14. We have applied these methods for retrospective screening of archived paraffin-embedded BM biopsies from PV patients.A cohort of 101 PV patients was screened for JAK2 exons 12 and 14 mutations in DNA isolated from paraffin-embedded tissues. All included patients had bone marrow trephine biopsies exhibiting features consistent with or suspicious for PV. In addition, 6 samples with previously confirmed exon12 mutation by allele-specific PCR were tested by this HRM method. DNA was isolated from paraffin-embedded tissues after deparaffinization process in graded alcohols and xylene. Briefly, deparaffinized samples were digested with proteinase K for 3days and DNA was isolated using the DNeasy Micro Kit (Qiagen, Valencia, CA). Following DNA isolation, samples were analyzed by HRM for JAK2 exons 12 and 14. Short exon-specific PCR amplicons (90-100 bp) were generated, which allowed the detection of the mutation in degradated material. All abnormal samples were bi-directionally sequenced. All 6 known exon 12 mutated samples were identified by HRM. Moreover, we repeatedly identified an abnormal exon 12 melting curve in 1/101 paraffin embedded samples. Sequencing results for this sample indicate the presence of a large (~32 bp) duplication. All 7 positive samples were confirmed by direct sequencing (see table 1). Interestingly, all three delH538K539 insL samples identified used a distinct codon for L and also had reproducibly different melting profiles.Ninety-two of 101 patients were positive for JAK2V617F mutation. One patient sample exhibited an abnormal HRM profile from wild-type and V617F positive control analysis. Two mutations were found in this patient by direct sequencing including JAK2V617F mutation and a previously unreported mutation in exon 14 (L611S) and we are now determining if these two separate JAK2 mutations are in cis or trans.In summary, we developed an HRM assay suitable for detection JAK2 exons 12 and exon14 mutations in archival material. This method is suitable for routine laboratory detection of these mutations as well as screening of archived biological material for additional mutations. We report one case of JAK2V617F-positive PV presenting with an additional JAK2L611S.Table (1)SampleExon 12Mutation1delH538K539 insL2delH538K539 insL3delH538K539 insL4K539L5H538-K539delinsF6R541-E543delinsK732 bp duplicationThis work has been supported by the MPD-RC consortium (J.P.) and a “UICC International Cancer Technology Transfer Fellowship“ awarded to Dr. Burjanivova. TB, RN and MES contributed equally to this work. Disclosures:No relevant conflicts of interest to declare.

Reliable Gene Expression Measurements from Fine Needle Aspirates of Pancreatic Tumors: Effect of Amplicon Length and Quality Assessment

Biomarker use for pancreatic cancer diagnosis has been impaired by a lack of samples suitable for reliable quantitative RT-PCR (qRT-PCR). Fine needle aspirates (FNAs) from pancreatic masses were studied to define potential causes of RNA degradation and develop methods for accurately measuring gene expression. Samples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phosphate dehydrogenase, and effects on qRT-PCR were determined by using a 150-bp and a 80-bp amplicon for RPS6. Potential causes of and methods to circumvent RNA degradation were studied by using FNAs from a pancreatic cancer xenograft. RNA extracted from pancreatic mass FNAs was extensively degraded. Fragmentation was related to needle bore diameter and could not be overcome by alterations in aspiration technique. Multiplex PCR for glyceraldehyde-3-phosphate dehydrogenase could distinguish samples that were suitable for qRT-PCR. The use of short PCR amplicons (<100 bp) provided reliable gene expression analysis from FNAs. When appropriate samples were used, the assay was highly reproducible for gene copy number with minimal (0.0003 or about 0.7% of total) variance. The degraded properties of endoscopic FNAs markedly affect the accuracy of gene expression measurements. Our novel approach to designate specimens "informative" for qRT-PCR allowed accurate molecular assessment for the diagnosis of pancreatic diseases.

Detection of Increased Amounts of Cell-Free Fetal DNA with Short PCR Amplicons

A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA. We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp). Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons. The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.

AFLP-DERIVED METHODS AS A TOOL FOR STUDY OF GENOMIC, EPIGENOMIC AND TRANSCRIPTOMIC CHANGES IN STRESSED GRAPEVINE PLANTS

Many studies describing phenotypic and genetic variation among standard and stressed plants were already published. A possibility to estimate stress-induced changes in genome, epigenome and transcriptome arose by establishing three AFLP-based methods within our laboratory (standard AFLP, methylation-sensitive AFLP and cDNA-AFLP). Three variants of grapevine plants after different stress actions (stress by in vitro cultivation, in vitro thermotherapy and virus infection) were prepared for this study. In case of standard AFLP application, comparison of obtained spectra via 0/1 binary matrix is usually performed. Within our group of variants the results show insignificant changes in its genomes. MS-AFLP spectra evaluation needs more detailed analysis of results to extract more precise information about DNA methylation characters and impact of individual stress factors on epigenetic changes. From the point of view of functional methodology, the cDNA-AFLP appeared as the most difficult to establish. During first experiments only short PCR amplicons (at most 150 bp) were generated. Further, the calculated similarity coefficients showed surprisingly low values. These facts signalised certain unspecific RNA degradation before its conversion onto ds cDNA. The significant improvement of obtained results was recorded by using RNA later reagent, SpectrumTM Plant Total RNA Kit, RNase Out enzyme and MINT cDNA synthesis kit in cDNA-AFLP protocol. In the case of cDNA-AFLP spectra evaluation, it is possible either to prepare 0/1 binary matrix or differences can be estimated on the base of ratio between intensities of individual peaks. This fact should be taken into account if cDNA-AFLP results are compared with other fingerprinting methods as RAPD, SSR or standard AFLP.

Forensic genetic analysis of nine miniSTR loci in the Korean population

Nine miniSTR loci were analyzed in 191 unrelated individuals from Korea using three multiplex PCR systems (multiplex I: D1S1677, D2S441 and D4S2364; multiplex II: D10S1248, D14S1434 and D22S1045; multiplex III: D12S391, D16S3253 and D20S161). Due to the short PCR amplicons (<145 bp), miniSTR systems can effectively be used in forensic analysis with highly degraded DNAs. Allele frequencies and forensic parameters were calculated to evaluate their usefulness in forensic casework. The Exact Test demonstrated that all loci surveyed here were found to be no deviation from Hardy–Weinberg equilibrium, except two miniSTR markers (D4S2364 and D16S3253). When we compared the distribution of genetic variation of six miniSTR markers (D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045), the Exact Test revealed significant differences ( P < 0.05) between the Korean sample studied here and almost all of other samples of East Asian and European populations. The combined probability of match calculated from nine miniSTR loci was 1.28 × 10 −8, which is high degree of polymorphism. Thus, the miniSTR system, combined with other valuable miniSTR markers, may be suitable for recovering useful information in analyzing degraded DNA samples.

High resolution melting analysis of almond SNPs derived from ESTs

High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.

Consensus RT-nested PCR detection of yellow head complex genotypes in penaeid shrimp

A consensus RT-nested (n)PCR is described that detects the six distinct genotypic variants in the yellow head virus (YHV) complex. The PCR primers targeted ORF1b gene regions more highly conserved amongst the reference strains of YHV (genotype 1) and gill-associated virus (GAV, genotype 2) and a set of 57 field isolates containing multiple representatives of each genotype. The test employed short PCR (359 bp) and nPCR (147 bp) amplicons to minimise the effects of RNA degradation. To ensure ≤8-primer degeneracy, two primers were designed to each site, one accommodating sequence variations amongst genotype 1 isolates and the other variations amongst isolates of the other genotypes. The analytical sensitivity limits of the PCR and nPCR were estimated to be ∼1250 and ∼1.25 RNA copies, respectively. The superior group-specificity of the consensus RT-nPCR compared to other OIE-recommended PCR tests for YHV/GAV was demonstrated using RNA from 17 Penaeus monodon shrimp infected with representatives of each of the six genotypes. Phylogenetic analysis using the 94 nt ORF1b gene sequence spanned by the nPCR primers generated genotype assignments that were consistent with those obtained using the extended 671 nt sequence used for the initial identification of genotypes.

Feasibility of Transferring Fluorescent In Situ Hybridization Probes to an 18S rRNA Gene Phylochip and Mapping of Signal Intensities

DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.

High-throughput SNP detection using nano-scale engineered biomagnetite

A semi-automated system for the large-scale detection of single nucleotide polymorphisms (SNPs) has been developed based on allele-specific oligonucleotide hybridization and thermal dissociation curve analysis using nano-scale engineered biomagnetite (bacterial magnetic particles; BacMPs). For reliable detection in large numbers of samples, several conditions for the capture of target DNA on nano-sized BacMPs and the denaturation of double-stranded DNA were optimized. The most efficient target DNA capture was observed using short PCR amplicons (69 bp). Captured DNAs were denatured using 50 mM NaOH. With these optimizations, large-scale SNP detection was performed on 822 samples of the transforming growth factor (TGF)-β1 gene, which is rich in both GC content and repetitive sequences. High reliability for the semi-automated BacMP-based SNP detection system was confirmed following comparison to traditional sequencing-based methods.

Targeted Gene-Expression Analysis by Genome-Controlled Reverse Transcription-PCR

For gene-expression analysis, which is anticipated to play an important role in classification of tumors and premalignant conditions, PCR-based quantitative assays must have increased diagnostic quantitative accuracy and reproducibility and enable analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples. We developed a reverse transcription-PCR-based quantitative assay that modifies the cDNA sequence to increase the melting temperature of short (56-64 bp) PCR amplicons, enabling their quantification in-tube by homogeneous melting-curve analysis. We used this method to analyze the expression of 8 genes, 7 potential colon cancer markers, and 1 control in samples obtained from 3 colon carcinoma cell lines, endoscopic biopsy from 8 patients undergoing gastroscopy for Barrett esophagus, and archival FFPE and frozen tissue from 20 patients who underwent surgery for colon carcinoma. The detection limit of the assay, when optimized for FFPE samples, was 100 copies of cDNA, and the dynamic range was 3 orders of magnitude. A prototype assay containing a panel of 8 genes displayed good reproducibility compared with the commercially available TaqMan assay (interassay CVs, 5%-20% vs 7%-43%, respectively). Gene-expression analysis was performed successfully in 26 (96%) of 27 endoscopic biopsy specimens, 30 (86%) of 35 archival FFPE samples, and 20 (100%) of 20 archival frozen samples. This new technology combines the reproducibility of competitive PCR with accurate quantitative detection by in-tube melting-curve analysis, enabling efficient analysis of mRNA profiles in samples with small numbers of cells or small amounts of tissue, as well as in archival FFPE tissues.

Effects of Target Length on the Hybridization Efficiency and Specificity of rRNA-Based Oligonucleotide Microarrays

The effect of target size on microarray hybridization efficiencies and specificity was investigated using a set of 166 oligonucleotide probes targeting the 16S rRNA gene of Escherichia coli. The targets included unfragmented native rRNA, fragmented rRNA ( approximately 20 to 100 bp), PCR amplicons (93 to 1,480 bp), and three synthetic single-stranded DNA oligonucleotides (45 to 56 bp). Fluorescence intensities of probes hybridized with targets were categorized into classes I (81 to 100% relative to the control probe), II (61 to 80%), III (41 to 60%), IV (21 to 40%), V (6 to 20%), and VI (0 to 5%). Good hybridization efficiency was defined for those probes conferring intensities in classes I to IV; those in classes V and VI were regarded as weak and false-negative signals, respectively. Using unfragmented native rRNA, 13.9% of the probes had fluorescence intensities in classes I to IV, whereas the majority (57.8%) exhibited false-negative signals. Similar trends were observed for the 1,480-bp PCR amplicon (6.6% of the probes were in classes I to IV). In contrast, after hybridization of fragmented rRNA, the percentage of probes in classes I to IV rose to 83.1%. Likewise, when DNA target sizes were reduced from 1,480 bp to 45 bp, this percentage increased approximately 14-fold. Overall, microarray hybridization efficiencies and specificity were improved with fragmented rRNA (20 to 100 bp), short PCR amplicons (<150 bp), and synthetic targets (45 to 56 bp). Such an understanding is important to the application of DNA microarray technology in microbial community studies.