Human gDNA Research Articles

The “COLD-PCR Approach” for Early and Cost-Effective Detection of Tyrosine Kinase Inhibitor Resistance Mutations in EGFR-Positive Non–Small Cell Lung Cancer

Activating epidermal growth factor receptor (EGFR) gene mutations can be successfully treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs), but nearly 50% of all patients' exhibit progression of the disease until treatment because of T790M mutations. It is proposed that this is mostly caused by therapy-resistant tumor clones harboring a T790M mutation. Until now no cost-effective routine-diagnostic method for EGFR-resistance mutation status analysis is available leaving long-time response to TKI treatment to chance. Unambiguous identification of T790M EGFR mutations is mandatory to optimize initial treatment strategies. Artificial EGFR T790M mutations and human wild-type gDNA were prepared in several dilution series. Preferential amplification using coamplification at lower denaturation temperature-PCR (COLD-PCR) of the mutant sequence and subsequent HybProbe melting curve detection or pyrosequencing were performed in comparison to normal processing. COLD-PCR-based amplification allowed the detection of 0.125% T790M mutant DNA in a background of wild-type DNA in comparison to 5% while normal processing. These results were reproducible. COLD-PCR is a powerful and cost-effective tool for routine diagnostic to detect underrepresented tumor clones in clinical samples. A diagnostic tool for unambiguous identification of T790M-mutated minor tumor clones is now available enabling optimized therapy.

The potential of the human immune system to develop broadly neutralizing HIV-1 antibodies

Objectives and design:Developing an effective HIV-1 vaccine that elicits broadly neutralizing HIV-1 human antibodies (bnAbs) remains a challenging goal. Extensive studies on HIV-1 have revealed various strategies employed by the virus to escape host immune surveillance. Here, we investigated the human antibody gene repertoires of uninfected and HIV-1-infected individuals at genomic DNA (gDNA) and cDNA levels by deep sequencing followed by high-throughput sequence analysis to determine the frequencies of putative germline antibody genes of known HIV-1 monoclonal bnAbs (bnmAbs).Methods:Combinatorial gDNA and cDNA antibody libraries were constructed using the gDNAs and mRNAs isolated from uninfected and HIV-1-infected human peripheral blood mononuclear cells (PBMCs). All libraries were deep sequenced and sequences analysed using IMGT/HighV-QUEST software (http://imgt.org/HighV-QUEST/index.action). The frequencies of putative germline antibodies of known bnmAbs in the gDNA and cDNA libraries were determined.Results and conclusion:The human gDNA antibody libraries were more diverse in heavy and light chain V-gene lineage usage than the cDNA libraries, indicating that the human gDNA antibody gene repertoires may have more potential than the cDNA repertoires to develop HIV-1 bnAbs. The frequencies of the heavy and kappa and lambda light chain variable regions with identical V(D)J recombinations to known HIV-1 bnmAbs were extremely low in human antibody gene repertoires. However, we found relatively high frequencies of the heavy and kappa and lambda light chain variable regions that used the same V-genes and had the same CDR3 lengths as known HIV-1 bnmAbs regardless of (D)J-gene usage. B-cells bearing B-cell receptors of such heavy and kappa and lambda light chain variable regions may be stimulated to induce HIV-1 bnAbs.

Multiply-primed rolling circle amplification of human papillomavirus using sequence-specific primers

Multiply-primed rolling circle amplification (RCA) is a suitable technique for amplification of circular templates and has been used to identify novel human papillomaviruses (HPV). In this study we develop an efficient RCA for whole genome amplification of HPV using HPV-specific primers in clinical samples and establish a protocol for whole genome sequencing using the Sanger method. Amplification of cloned HPV-genomes by RCA was compared using specific primers against random hexamers. Using HPV-specific primers increased the effectiveness on average 15.2 times and the enrichment of HPV relative to human gDNA on average 62.2 times, as compared to using random hexamer. RCA products were sequenced without need for cloning, even when using low-input amounts. The technique was successfully used on 4 patient samples from FTA cards, to generate whole HPV-genome sequences. Degenerated HPV-specific primers for RCA produce DNA of sufficient quality and quantity suitable for sequencing and other potential downstream analyses.

HMLH1Gene is not Methylated in Osteosarcoma

Background: Epigenetic silencing of the hMLH1 gene by promoter methylation plays a role in initiation and progression of cancers by leading to increased genomic instability. There is no report on methylation status of the hMLH1 gene in osteosarcoma. Methods: In this study, we aimed to determine the methylation status of the hMLH1 gene in human osteosarcoma cells. The methylation status of the promoter region of the hMLH1 gene was determined by bi-sulfite sequencing (BSS) in U2OS human osteosarcoma and normal human diploid fibroblast cells (NHDFc). Methylated human gDNA by Sss I methylase enzyme was also used as a positive control. Results: Methylation was not detected in the promoter of the hMLH1 gene—neither in U2OS nor in NHDF cells. Conclusions: Our data showed the hMLH1 gene is not methylated in osteosarcoma, which may imply the genomic instability caused by hMLH1 inactivation does not have a role in osteosarcomas, unlike most other tumors.

Detection of specific DNA using a microfluidic device featuring tethered poly(N-isopropylacrylamide) on a silicon substrate

In this study, we grafted thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) onto a Si substrate as the medium in a microfluidic device to detect specific DNA molecules [human genomic DNA (hgDNA528), 528 bp] at extremely low concentrations (down to 2 ng/μl). After using the polymerase chain reaction to amplify the released human gDNA signal from the tethered PNIPAAm on the substrate, the amplified human gDNA molecules were characterized through agarose gel electrophoresis. The tethered PNIPAAm in the fluid device allowed the precise detection of the human gDNA.

A procedure for highly specific, sensitive, and unbiased whole-genome amplification

Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a method using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to subfemtograms of DNA. With an input of as little as 0.5-2.5 ng of human gDNA or a few cells, the product could be close to native DNA in locus representation. The amplicons from 5 and 0.5 ng of DNA faithfully demonstrated all previously known heterozygous segmental duplications and deletions (3 Mb to 18 kb) located on chromosome 22 and even a homozygous deletion smaller than 1 kb with high-resolution chromosome-wide comparative genomic hybridization. With 550k Infinium BeadChip SNP typing, the >99.7% accuracy was compared favorably with results on unamplified DNA. Importantly, underrepresentation of chromosome termini that occurred with GenomiPhi v2 was greatly rescued with the present procedure, and the call rate and accuracy of SNP typing were also improved for the amplicons with a 0.5-ng, partially degraded DNA input. In addition, the amplification proceeded logarithmically in terms of total yield before saturation; the intact cells was amplified >50 times more efficiently than an equivalent amount of extracted DNA; and the locus imbalance for amplicons with 0.1 ng or lower input of DNA was variable, whereas for higher input it was largely reproducible. This procedure facilitates genomic analysis with single cells or other traces of DNA, and generates products suitable for analysis by massively parallel sequencing as well as microarray hybridization.

Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform

This study presents a new magnetic bead-based microfluidic platform, which integrates three major modules for rapid leukocytes purification, genomic DNA (gDNA) extraction and fast analysis of genetic gene. By utilizing microfluidic technologies and magnetic beads conjugated with CD15/45 antibodies, leukocytes in a human whole blood sample can be first purified and concentrated, followed by extraction of gDNA utilizing surface-charge switchable, DNA-specific, magnetic beads in the lysis solution. Then, specific genes associated with genetic diseases can be amplified by an on-chip polymerase chain reaction (PCR) process automatically. The whole pretreatment process including the leukocytes purification and gDNA extraction can be performed in an automatic fashion with the incorporation of the built bio-separators consisting of microcoils array within less than 20 min. The detection of single nucleotide polymorphism (SNP) genotyping of methylenetetra-hydrofolate reductase (MTHFR) C677T region associated with an increased risk of genetic diseases was further performed to demonstrate the capability of the proposed system. The extracted gDNA can be transported into a micro PCR chamber for on-chip fast nucleic acid amplification of detection genes with minimum human intervention. Hence, the developed system may provide a powerful automated platform for pretreatment of human leukocytes, gDNA extraction and fast analysis of genetic gene.

Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g™ WGA using gDNA from two paired cytobrushes (cytobush ‘A’ kept in a cell lysis buffer, and ‘B’ dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.

Establishment of the 1st International Genetic Reference Panel for Factor V Leiden, human gDNA

Forty-one laboratories participated in an international collaborative study to assess the suitability of a panel of three genomic DNA samples as the 1st International Genetic Reference Panel for the Factor V Leiden (FVL) variant. The code numbers of the materials were 03/254 (FV wild type), 03/260 (FVL homozygote) and 03/248 (FVL heterozygote). The participants evaluated the panel against their in-house controls which were known patient samples and commercial controls. In total, 859 genotype tests were carried out on the panel, with an error rate of 0.7%. The errors were not related to specific samples of the panel or to any specific techniques. The findings of this study have indicated that this panel is suitable to be used as a reference material for genotyping of factor V Leiden. It was therefore recommended that the three genomic DNA samples be established as the 1st International Genetic Reference Panel for Factor V Leiden, Human gDNA, 04/224. This recommendation was approved by the Scientific and Standardization Committee (SSC) of the ISTH (International Society on Thrombosis and Haemostasis) in June 2004 and the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) in November 2004.

Identification of a meander region proline residue critical for heme binding to cytochrome P450: implications for the catalytic function of human CYP4B1.

Alignment of xenobiotic-metabolizing P450 protein sequences highlights an invariant proline residue in the meander region two amino acids N-terminal to the distal arginine of the putative ERR triad thought to be important for heme binding. This occurs as a serine in the sequences derived from human CYP4B1 gDNA and both human lung and placental CYP4B1 cDNAs. Reversion of this serine to the conserved proline residue (Ser427 --> Pro) by site-directed mutagenesis conferred the ability to incorporate heme on the human placental enzyme. Mutation of the corresponding proline in rabbit CYP4B1 (Pro422 --> Ser) abolished heme incorporation. Membrane preparations of human CYP4B1(Pro) and rabbit CYP4B1(Pro), but not the corresponding CYP4B1(Ser) variants, supported lauric acid hydroxylation preferentially at the omega-position. Purified, reconstituted human CYP4B1(Pro) and rabbit CYP4B1(Pro) formed 12-hydroxylauric acid at rates of 17-21 min-1, and both enzymes were also C-8 to C-10 fatty acid omega-hydroxylases preferentially, with total rates of hydroxylation decreasing in the order C-12 > C-10 > C-9 > C-8. Finally, neither human nor rabbit CYP4B1(Pro) formed detectable levels of any hydroxylated testosterone metabolites. Therefore, the presence of a consensus Pro-X-Arg motif is critical for incorporation of the heme prosthetic group in human and rabbit CYP4B1 proteins expressed in insect cells. Native human CYP4B1, expressed in vivo, is likely to be functionally impaired if Pro427 is required for holoenzyme expression in mammalian cells.