Degenerate Oligonucleotide PCR Research Articles

Dynamic expression and steroid regulation of sarg, a novel gene in the perinatal lung

BackgroundThe fetal lung's preparation for birth consists of structural and physiologic maturation processes associated with underlying changes in gene expression. Understanding this gene expression program may help to develop novel...

Abstract 1160: Whole genome amplification and high-throughput sequencing of formalin-fixed paraffin-embedded colorectal cancer

Abstract Formalin-fixed paraffin-embedded (FFPE) tumors represent a valuable resource for translational cancer genomic research. However, genetic analysis of the FFPE material is challenging, as archived tissues are often small biopsies and the fixation process damages DNA. In researching a solution to this, we have evaluated the performance of different whole genome amplification methods for obtaining DNA samples from archived tumors and their use as a template in high-throughput sequencing. Two whole genome amplification methods were applied using 20 μm sections of FFPE colorectal cancer tissue and normal human genome DNA samples. Genomic DNA was amplified using Multiple Displacement Amplification (MDA) method and a modified version of Degenerate Oligonucleotide PCR (DOP) assay. Non-amplified human genomic DNA was used as a reference when genomic coverage, linearity and uniformity of the amplified products were assessed. Paired-end sequencing libraries were prepared from the DOP amplified templates while the MDA samples underwent single read library preparation. Sequencing was performed using an Illumina Genome Analyzer II. 2.5 million 54-mer reads mapping to the human genome were generated for each sample. Whole genome amplification was shown to produce artifacts when compared to non-amplified genomic DNA sequences. The modified DOP method preferentially amplified non-repetitive sequences in the human genome. Using the MDA-based method, genomic sequence coverage was generally more even between regions of repetitive and non-repetitive DNA. However, spread of the sequence read counts was greater in the MDA method and MDA amplification offered less dynamic range to identify copy number aberrations. Using whole genome amplification methods with the FFPE colorectal tumor sample we were able to generate 130 Mb of genomic sequence data and identify DNA copy number gains in chromosomes 13 and 20q. Whole genome amplification and high-throughput sequencing can be used to generate sequence data from minute amounts of FFPE material and identify DNA copy number aberrations in the archived tumors. Nonetheless, whole genome amplification produces artifacts by introducing bias towards specific regions of the genome, generating artificial and random DNA fragment assemblies (MDA) and introducing universal primer sequence in the ends of the amplified DNA fragments (DOP). 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 1160.

Construction and use of spotted large-insert clone DNA microarrays for the detection of genomic copy number changes

Microarray-based comparative genomic hybridization has become a widespread method for the analysis of DNA copy number changes across the human genome. Initial methods for microarray construction using large-insert clones required the preparation of DNA from large-scale cultures. This rapidly became an expensive and time-consuming process when expanded to the number of clones needed for higher resolution arrays. To overcome this problem, several PCR-based strategies have been developed to enable array construction from small amounts of cloned DNA. Here, we describe the construction of microarrays composed of human-specific large-insert clones (40-200 kb) using a specific degenerate oligonucleotide PCR strategy. In addition, we also describe array hybridization using manual and automated procedures and methods for array analysis. The technology and protocols described in this article can easily be adapted for other species dependent on the availability of clone libraries. According to our protocols, the procedure will take approximately 3 days from labeling the DNA to scanning the hybridized slides.

Activation of prostate-specific antigen precursor (pro-PSA) by prostin, a novel human prostatic serine protease identified by degenerate PCR.

A novel serine protease was found in human prostate by degenerate oligonucleotide PCR amplification and cloned. The zymogen form of this enzyme, named prostinogen, is composed of 240 amino acid residues with an amino-terminal propiece of 5 residues and a 235-residue mature enzyme. The transcript has a signal peptide of 15 amino acid residues. The mature enzyme has 41% sequence identity with prostate specific antigen (PSA). Prostinogen was expressed in Escherichia coli and refolded from inclusion bodies. The zymogen, with a molecular mass of 28 kDa, was readily activated by agarose-immobilized trypsin to generate prostin, a serine protease, which cleaves the chromogenic substrate (N-benzoyl-L-Ile-L-Glu-L-Gly-L-Arg-p-nitroaniline hydrochloride) (S-2222). Recombinant prostin readily activates the precursor of PSA (pro-PSA) by cleavage of the amino terminal Arg(7)-Ile(8) peptide bond. These results indicate that prostin may be a physiological activator of pro-PSA following its own proteolytic activation, as part of a cascade system involving a series of serine protease precursor proteins in the prostate.

Cloning and primary structure of putative cytosolic and mitochondrial malate dehydrogenase from the mollusc Nucella lapillus (L.).

The evolutionary history of the malate dehydrogenase (MDH) gene family [NAD-dependent MDH; EC 1.1.1.37 and NAD(P)-dependent MDH; EC 1.1.1.82] has received much attention. MDHs have also featured extensively as electrophoretic markers in population genetics and evolutionary ecology, and in many cases, intraspecific variation in MDH has been correlated with environmental variables. However, while the amino acid residues essential for MDH function are known, no studies have examined intraspecific nucleotide variation despite evidence indicating that natural selection may be operating on this locus. This study presents two sets of degenerate oligonucleotide PCR primers to facilitate the cloning of cytosolic MDH (cMDH) and mitochondrial MDH (mMDH) from a broad range of animals (cMDH) and animals and plants (mMDH). These primers were used to obtain putative cMDH and mMDH cDNAs from the mollusc Nucella lapillus. The N. lapillus cMDH cDNA was found to encode a putative cMDH protein of 334aa and 36kDa, while the mMDH cDNA encoded a putative mature mMDH protein of 315aa and 33kDa. The putative amino acid sequences of the two compartmentalised N. lapillus MDHs are presented and compared to other known MDH sequences.

Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis.

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29-53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.

Mosquito transferrin, an acute-phase protein that is up-regulated upon infection.

When treated with heat-killed bacterial cells, mosquito cells in culture respond by up-regulating several proteins. Among these is a 66-kDa protein (p66) that is secreted from cells derived from both Aedes aegypti and Aedes albopictus. p66 was degraded by proteolysis and gave a virtually identical pattern of peptide products for each mosquito species. The sequence of one peptide (31 amino acids) was determined and found to have similarity to insect transferrins. By using conserved regions of insect transferrin sequences, degenerate oligonucleotide PCR primers were designed and used to isolate a cDNA clone encoding an A. aegypti transferrin. The encoded protein contained a signal sequence that, when cleaved, would yield a mature protein of 68 kDa. It contained the 31-amino acid peptide, and the 3' end exactly matched a cDNA encoding a polypeptide that is up-regulated when A. aegypti encapsulates filarial worms [Beerntsen, B. T., Severson, D. W. & Christensen, B. M. (1994) Exp. Parasitol. 79, 312-321]. This transferrin, like those of two other insect species, has conserved iron-binding residues in the N-terminal lobe but not in the C-terminal lobe, which also has large deletions in the polypeptide chain, compared with transferrins with functional C-terminal lobes. The hypothesis is developed that this transferrin plays a role similar to vertebrate lactoferrin in sequestering iron from invading organisms and that degradation of the structure of the C-terminal lobe might be a mechanism for evading pathogens that elaborate transferrin receptors to tap sequestered iron.

Identification and characterization of the tolQRA genes of Pseudomonas aeruginosa.

The tolQ, tolR, and tolA genes from Pseudomonas aeruginosa PAO were cloned using degenerate oligonucleotide PCR primers designed based on conserved transmembrane regions of Escherichia coli TolQ and TolR and E. coli and Pseudomonas putida ExbB and ExbD. The resulting PCR product was used as a probe to isolate a 6.5-kb DNA fragment containing P. aeruginosa tolQ, tolR, and tolA. The nucleotide sequence of a 2.9-kb DNA fragment containing the tolQ, tolR, and tolA genes was determined. The DNA sequence predicts TolQ to be a 25,250-Da protein exhibiting 53% identity to E. coli TolQ. TolR is predicted to be a 15,788-Da protein, sharing 38% identity with the E. coli TolR protein. The P. aeruginosa tolA sequence predicts a 37,813-Da protein with 27% identity to the E. coli TolA. The P. aeruginosa TolQRA proteins were expressed in E. coli minicells. Analysis of plasmid-encoded tolQ::lacZ and tolA::lacZ promoter fusions in E. coli indicated that these genes are expressed at different levels, suggesting transcription from different promoters. Transcriptional analysis of the tol genes in P. aeruginosa revealed that the tolQ and tolR genes are cotranscribed as an approximately 1.5-kb transcript and that tolA is transcribed from its own promoter as an approximately 1.2-kb transcript. The P. aeruginosa Tol proteins were functionally unable to complement E. coli tol mutants, although P. aeruginosa TolQ was able to complement the iron-limited growth of an E. coli exbB mutant. Introduction of the tolQRA genes in the tol-like mutant PAO 1652 restored pyocin AR41 killing, indicating that the Tol proteins are involved in the uptake of pyocin AR41 in P. aeruginosa. Attempts to inactivate the chromosomal copy of the tolA or tolQ gene in the parent strain PAO proved to be unsuccessful, and we propose that inactivation of these genes in P. aeruginosa results in a lethal phenotype.

Identification of conserved nucleotide sequences within the GB virus C 5′-untranslated region: Design of PCR primers for detection of viral RNA

Recently, the discovery of a new human RNA virus, GB virus C (GBV-C), was reported. GBV-C was isolated from the serum of a West African individual using degenerate oligonucleotide PCR primers designed from a consensus sequence of the NS3 helicase genes of hepatitis C virus (HCV), GBV-A, and GBV-B. Seven other individuals were shown to be infected with GBV-C via RT-PCR using these primers. Subsequently, degenerate PCR primers based upon a consensus sequence of the eight original isolates were designed. These primers were shown to be superior to the original set. However, since they were derived from a region of the viral genome exhibiting up to 17% nucleotide sequence divergence, mismatch between the primers and template may result in an underestimation of the true GBV-C prevalence. To overcome this potential problem, we obtained the sequences at the 5'-untranslated region (UTR) of the GBV-C genome from 35 infected individuals and identified regions of high sequence conservation among the isolates. We describe the design and testing of PCR primers derived from conserved sequences within the 5'-UTR of the GBV-C genome. These primers were shown to be as effective as the helicase-derived primers in detecting GBV-C RNA in human sera.

A new non-LTR retrotransposon provides evidence for multiple distinct site-specific elements in Crithidia fasciculata miniexon arrays.

We have identified a new member of the family of trypanosome site-specific retrotransposons, using a degenerate oligonucleotide PCR strategy. The 9595 bp element, termed Crithidia retrotransposable element 2 (CRE2), was cloned and found to be inserted in the tandemly arrayed miniexon genes of Crithidia fasciculata. The element is flanked by 29 bp target site duplications but lacks the 3' poly dA tract characteristic of most other non-long terminal repeat retrotransposons. The amino terminal region of the single 2518-codon open reading frame contains a putative metal-binding motif and a proline-rich region similar to gag-like domains of other retrotransposons. The carboxy terminal region of this open reading frame shares sequence homology with the reverse transcriptase and putative endonuclease regions of three previously described trypanosomatid site-specific retrotransposons. All four of these retrotransposons are specifically inserted between nucleotides 11 and 12 of the highly conserved 39mer sequence of the miniexon gene. Most copies of CRE2 and the previously characterized CRE1 are located on different sized chromosomes. Additional CRE-related sequences were identified by screening Crithidia libraries. These results suggest that a particular sequence in the C. fasciculata miniexon repeat is the target for multiple distinct site-specific retrotransposon insertions.