Whole genome amplification (WGA) is a practical solution to eliminate molecular analysis limitations associated with genomic DNA (gDNA) quantity. Different methods have been developed to amplify the whole genome, including primer extension preamplification (PEP), degenerate oligonucleotide primed PCR (DOP-PCR), and multiple displacement amplification (MDA). Each of these methods has its own merits and limitations.
Effects of primer length and composition on amplification quality and quantity were explored in this study at two different temperatures (30°C & 40°C). New primer designs combined with elevated amplification temperature has significantly improved MDA as measured by amplification yield, genome coverage, and allele drop out (ADO) analysis. A remarkable finding was the comprehensive amplification, at 30°C & 40°C, of the human whole genome via the use of GGGCAGGA*N*G hotspot recombination consensus primer. Amplification was characterized by Affymetrix 10K SNP chip analysis. Finally, the use of new primer designs has suppressed the template-independent DNA amplification (TIDA) both at 30°C and 40°C.
The use of new primers in this study combined with elevated incubation temperatures in MDA has remarkably improved the specificity, amplification yield, and suppressed TIDA.
To understand cancer progression, it is desirable to study the earliest stages of its development, which are often microscopic lesions. Array comparative genomic hybridization (aCGH) is a valuable high-throughput molecular approach for discovering DNA copy number changes; however, it requires a relatively large amount of DNA, which is difficult to obtain from microdissected lesions. Whole genome amplification (WGA) methods were developed to increase DNA quantity; however their reproducibility, fidelity, and suitability for formalin-fixed paraffin-embedded (FFPE) samples are questioned. Using aCGH analysis, we compared two widely used approaches for WGA: single cell comparative genomic hybridization protocol (SCOMP) and degenerate oligonucleotide primed PCR (DOP-PCR). Cancer cell line and microdissected FFPE breast cancer DNA samples were amplified by the two WGA methods and subjected to aCGH. The genomic profiles of amplified DNA were compared with those of non-amplified controls by four analytic methods and validated by quantitative PCR (Q-PCR). We found that SCOMP-amplified samples had close similarity to non-amplified controls with concordance rates close to those of reference tests, while DOP-amplified samples had a statistically significant amount of changes. SCOMP is able to amplify small amounts of DNA extracted from FFPE samples and provides quality of aCGH data similar to non-amplified samples.
Array Comparative Genomic Hybridisation (array CGH) is a powerful technique for the analysis of constitutional chromosomal anomalies. Chromosomal duplications or deletions detected by array CGH need subsequently to be validated by other methods. One method of validation is Fluorescence in situ Hybridisation (FISH). Traditionally, fluorophores or hapten labelling is performed by nick translation or random prime labelling of purified Bacterial Artificial Chromosome (BAC) products. However, since the array targets have been generated from Degenerate Oligonucleotide Primed (DOP) amplified BAC clones, we aimed to use these DOP amplified BAC clones as the basis of an automated FISH labelling protocol. Unfortunately, labelling of DOP amplified BAC clones by traditional labelling methods resulted in high levels of background.
We designed an improved labelling method, by means of degenerate oligonucleotides that resulted in optimal FISH probes with low background.
We generated an improved labelling method for FISH which enables the rapid generation of FISH probes without the need for isolating BAC DNA. We labelled about 900 clones with this method with a success rate of 97%.
Development of uncommon viral infections in immunocompromised transplant recipients can pose major diagnostic challenges. We present a case report of an immunocompromised patient suffering from pneumonia, for which the causative agent was not identified by routine methods.
To identify the potential cause of the pneumonia using a degenerate oligonucleotide primer (DOP) PCR assay which is designed to detect all viruses.
DOP-PCR was applied to bronchoalveolar lavage fluid from this patient. Generic PCR products were cloned and sequenced.
The novel universal virus assay detected human metapneumovirus in the clinical sample. The finding was confirmed by two independent metapneumovirus specific PCRs targeting independent regions of the viral genome.
The DOP PCR was used to detect and identify the sequence of an unidentified virus. This study provides proof of concept for the use on clinically relevant specimens of this unbiased universal assay, which requires no previous viral sequence information.
Virus discovery; virus detection; immunocompromised host; virus disease diagnosis; virus disease etiology
Intrahepatic cholangiocarcinoma (ICC), a malignant neoplasm of the biliary epithelium, is usually fatal because of difficulty in early diagnosis and lack of availability of effective therapy. The genetic mechanisms involved in the development of ICC are not well understood and only a few cytogenetic studies of ICC have been published. Recently, technique of degenerate oligonucleotide primed (DOP)-PCR comparative genomic hybridization (CGH) permits genetic imbalances screening of the entire genome using only small amounts of tumor DNA. In this study chromosomal aberrations in 33 Korean ICC were investigated by DOP-PCR CGH. The common sites of copy number increases were 20q (67%), 17 (61%), 11q11-q13 (42%), 8p12-qter (39%), 18p (39%), 15q22-qter (36%), 16p (36%), 6p21 (30%), 3q25-qter (27%), 1q41-qter (24%), and 5p14-q11.2 (24%). DNA amplification was identified in 16 carcinomas (48%). The frequent sites of amplification were 20q, 17p, 17q23-qter, and 7p. The most frequent sites of copy number decreases were 1p32-pter (21%) and 4q (21%). The recurrent chromosomal aberrations identified in this study provide candidate regions involved in the tumorigenesis and progression of ICC.
Cholangiocarcinoma, Chromosome Aberrations, Comparative Genomic Hybridization; Polymerase Chain Reaction
We have developed a method for direct selection of cDNAs using whole chromosomes as target DNA. Double-strand cDNAs were synthesized from human fetal brain polyadenylated mRNAs. Flow-sorted chromosomes 17 and 19 were amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and used to capture ds cDNAs by an improved magnetic bead capture protocol. To demonstrate the capabilities of this method, the selected cDNAs were used as probes in FISH experiments. The selected cDNA populations specifically painted chromosomes 17 or 19 on metaphase spreads. These results demonstrate that it is possible to do chromosome painting using cDNA probes and that this method is a means to rapidly select expressed sequences encoded by any portion of the genome.
Acute gastroenteritis (AGE) is a common illness affecting all age groups worldwide, causing an estimated three million deaths annually. Viruses such as rotavirus, adenovirus, and caliciviruses are a major cause of AGE, but in many patients a causal agent cannot be found despite extensive diagnostic testing. Proposing that novel viruses are the reason for this diagnostic gap, we used molecular screening to investigate a cluster of undiagnosed cases that were part of a larger case control study into the etiology of pediatric AGE. Degenerate oligonucleotide primed (DOP) PCR was used to non-specifically amplify viral DNA from fecal specimens. The amplified DNA was then cloned and sequenced for analysis. A novel virus was detected. Elucidation and analysis of the genome indicates it is a member of the Bocavirus genus of the Parvovirinae, 23% variant at the nucleotide level from its closest formally recognized relative, the Human Bocavirus (HBoV), and similar to the very recently proposed second species of Bocavirus (HBoV2). Fecal samples collected from case control pairs during 2001 for the AGE study were tested with a bocavirus-specific PCR, and HBoV2 (sequence confirmed) was detected in 32 of 186 cases with AGE (prevalence 17.2%) compared with only 15 controls (8.1%). In this same group of children, HBoV2 prevalence was exceeded only by rotavirus (39.2%) and astrovirus (21.5%) and was more prevalent than norovirus genogroup 2 (13.4%) and adenovirus (4.8%). In a univariate analysis of the matched pairs (McNemar's Test), the odds ratio for the association of AGE with HBoV2 infection was 2.6 (95% confidence interval 1.2–5.7); P = 0.007. During the course of this screening, a second novel bocavirus was detected which we have designated HBoV species 3 (HBoV3). The prevalence of HBoV3 was low (2.7%), and it was not associated with AGE. HBoV2 and HBoV3 are newly discovered bocaviruses, of which HBoV2 is the thirdmost-prevalent virus, after rotavirus and astrovirus, associated with pediatric AGE in this study.
Acute gastroenteritis (AGE) is a common illness affecting all age groups worldwide, causing an estimated three million deaths annually. However, in many patients a causal agent cannot be found despite extensive diagnostic testing. Proposing that novel viruses are the reason for this diagnostic gap, we screened fecal samples from symptomatic children using a molecular degenerate amplification technique and detected the presence of a novel parvovirus, Human Bocavirus species 2 (HBoV2). The genome of HBoV2 is 23% variant from its closest relative, the human bocavirus, a member of the Bocavirus genus of the Parvovirinae. Using specific amplification assays, we then found HBoV2 was the thirdmost-prevalent virus detected in samples from symptomatic children in a case control study of AGE. Further, we found virus presence was associated with symptoms. During this screening, we detected a second related parvovirus, which we have named Human Bocavirus species 3 (HBoV3), but the prevalence was low and not associated with symptoms. The discovery of HBoV2 has reduced the diagnostic gap, but more studies are required to further investigate its role in AGE.
Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, −2.1%, and −13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.
A simple and efficient method for the dissection of (marker) chromosomes, (micro)nuclei, and chromosome regions is presented. Before microdissection, metaphases are overlaid with milli-Q water to rehydrate the chromosomes, which makes them soft and sticky. The dissected chromosome fragments are dissolved without proteinase-K or topoisomerase treatment and directly amplified using a degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). The advantages of this microFISH method over previously reported methods are: (1) microdissection in this way is very fast; (2) a chromosome, marker, (micro)nucleus, or chromosome region is collected as a whole using only one microneedle; (3) the dissected material sticks tightly to the needle without the risk of getting lost; (4) no Sequenase is used in the DOP-PCR reaction which reduces the risk of contamination.
To investigate the distribution of tumour cells expressing the SYT–SSX fusion gene in biphasic synovial sarcoma, modified reverse transcription polymerase chain reaction (RT–PCR) analysis was performed using microdissected specimens from haematoxylin and eosin stained sections of archival paraffin wax embedded tissues. This modified RT–PCR included a stage with degenerate oligonucleotide primed (DOP) PCR, which randomly amplified cDNA after reverse transcription. SYT–SSX fusion transcripts were detected in both epithelial and spindle cell areas of all three biphasic synovial sarcomas examined. Subsequent sequence analysis confirmed that the detected messages were derived from the SYT–SSX1 fusion gene in two cases and from SYT–SSX2 in one. These results indicate that SYT–SSX fusion transcripts are found in both epithelial and spindle cell areas of biphasic synovial sarcoma, and RT–DOP–PCR–PCR analysis is a useful method for detection of extremely small amounts of mRNA in microdissected samples from archival formalin fixed, paraffin wax embedded tumour tissues.
biphasic synovial sarcoma; SYT-SSX fusion transcript; degenerated oligonucleotide primed polymerase chain reaction; laser capture microdissection
Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR) from the ovarian cancer cell line UACC-1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co-amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo-generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo-generated small palindromic sequences, which may be generated through non-homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification.
gene amplification; double minutes; junction sequence; amplicon boundary palindrome; cancer
Cytogenetic analysis of a 4 year old girl with developmental delay and dysmorphic features showed extra chromosomal material of unknown origin on 20p (46,XX,add(20)(p13)). Familial chromosome studies showed direct inheritance of add(20)(p13) from the father, who had a similar, albeit milder, phenotype. Fibroblast chromosome studies of the father showed no karyotype mosaicism. The additional material could not be identified on the basis of the G banding pattern owing to its small size and ambiguous banding pattern. Chromosome microdissection of the unknown material was performed, the DNA was amplified and labelled using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and reverse painted to the proband's cells to show the karyotype 46,XX,der(20)t(6;20)(p23;p13), conferring partial trisomy 6p and presumed partial monosomy for 20p. Chromosome microdissection has made possible the first reported case of directly inherited partial trisomy 6p.
Keywords: partial trisomy; chromosome 6p; microdissection; duplication
Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs) or large scale (CGH array, FISH) methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls.
All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR.
Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.
Whole genome approaches using single nucleotide polymorphism (SNP) markers have the
potential to transform complex disease genetics and expedite pharmacogenetics research.
This has led to a requirement for high-throughput SNP genotyping platforms.
Development of a successful high-throughput genotyping platform depends on coupling
reliable assay chemistry with an appropriate detection system to maximise efficiency with
respect to accuracy, speed and cost. Current technology platforms are able to deliver
throughputs in excess of 100 000 genotypes per day, with an accuracy of >99%, at a cost
of 20–30 cents per genotype. In order to meet the demands of the coming years, however,
genotyping platforms need to deliver throughputs in the order of one million genotypes per
day at a cost of only a few cents per genotype. In addition, DNA template requirements
must be minimised such that hundreds of thousands of SNPs can be interrogated using a
relatively small amount of genomic DNA. As such, it is predicted that the next generation
of high-throughput genotyping platforms will exploit large-scale multiplex reactions and
solid phase assay detection systems.
Microsatellite (SSR) and single nucleotide polymorphism (SNP) markers are widely used in plant breeding and genomic research. Thus, methods to improve the speed and efficiency of SSR and SNP genotyping are highly desirable. Here we describe a new method for multiplex PCR that facilitates fluorescence-based SSR genotyping and the multiplexed preparation of DNA templates for SNP assays.
We show that multiplex-ready PCR can achieve a high (92%) success rate for the amplification of published sequences under standardised reaction conditions, with a PCR specificity comparable to that of conventional PCR methods. We also demonstrate that multiplex-ready PCR supports an improved level of multiplexing in plant genomes of varying size and ploidy, without the need to carefully optimize assay conditions. Several advantages of multiplex-ready PCR for SSR and SNP genotyping are demonstrated and discussed. These include the uniform amplification of target sequences within multiplexed reactions and between independent assays, and the ability to label amplicons during PCR with specialised moieties such fluorescent dyes and biotin.
Multiplex-ready PCR provides several technological advantages that can facilitate fluorescence-based SSR genotyping and the multiplexed preparation of DNA templates for SNP assays. These advantages can be captured at several points in the genotyping process, and offer considerable cost and labour savings. Multiplex-ready PCR is broadly applicable to plant genomics and marker assisted breeding, and should be transferable to any animal or plant species.
A new method for DNA diagnostics based on template-directed primer extension and detection by fluorescence resonance energy transfer is described. In this method, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5'-fluorescein-labeled primer (designed to hybridize to the DNA template adjacent to the polymorphic site) in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq DNA polymerase (Klentaq1-FY). The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification. This homogeneous DNA diagnostic method, which we call the template-directed dye-terminator incorporation assay, is shown to be highly sensitive and specific and is suitable for automated genotyping of large numbers of samples.
By random amplification of a microdissected chromosome using the degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and forward painting (microFISH), we characterised an extra structurally abnormal chromosome (ESAC) or supernumerary marker chromosome in a mentally retarded girl with a pattern of dysmorphic features. It could be clearly shown that the small marker chromosome originates from two different regions of chromosome 18, 18p11.1→18q11.1 and 18q12.3→18q21.1 respectively. Maternal origin of the de novo ESAC and biparental origin of the normal homologues of chromosome 18 were shown by PCR of several highly polymorphic microsatellites. In this case, application of microFISH was a prerequisite for rapid and precise characterisation of an ESAC. A definite identification of this discontinuous supernumerary marker chromosome would not have been possible using FISH with centromere specific probes or multicolour FISH approaches.
Keywords: supernumerary marker chromosome; microdissection; FISH; chromosome 18
We describe a method, termed reverse chromosome painting, which allows the rapid analysis of the content and breakpoints of aberrant chromosomes. The method involves the sorting of small numbers of the aberrant chromosome from short term blood culture preparations or cell lines by using bivariate flow karyotype analysis. The sorted chromosomes are amplified and biotin labelled enzymatically using a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), the product annealed to metaphase spreads from normal subjects, and hybridisation detected using fluorescence in situ hybridisation (FISH). We show the usefulness of this method for routine clinical cytogenetics by the analysis of cases involving an insertion, a deletion, a translocation, and two cases of a chromosome with additional material of unknown origin. The method has particular application for the rapid resolution of the origin of de novo unbalanced chromosome duplications.
We have previously described a fluorescence in situ hybridisation (FISH) assay for the simultaneous analysis of all human subtelomeric regions using a single microscope slide. Here we report the use of this multiprobe FISH assay in the study of a patient whose karyotype was reported by G banding analysis as 46,XX,del(18)(p11.2). Although the proband had some features suggestive of a chromosomal abnormality, relatively few of the specific features of del(18p) were present. She was a 37 year old female with mild distal spinal muscular atrophy (SMA), arthritis of the hands, an abnormal chest shape (pectus excavatum), and an unusual skin condition (keratosis pilaris). Reverse chromosome painting with degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) amplified del(18p) chromosomes as a probe confirmed the abnormality as del(18p), with no evidence of any other chromosome involvement. Subsequently, the multiprobe FISH assay confirmed deletion of 18p subtelomeric sequence. However, the assay also showed that sequences corresponding to the 2p subtelomeric probe were present on the tip of the shortened 18p. The patient is therefore monosomic for 18p11.2-pter and trisomic for 2p25-pter, and the revised karyotype is 46,XX,der(18)t(2;18)(p25; p11.2). We believe that a proportion of all cases reported as telomeric deletions may be cryptic translocations involving other chromosome subtelomeric regions. Further studies such as this are necessary to define accurately the clinical characteristics associated with pure monosomy in chromosomal deletion syndromes.
Chromosome specific comparative genome hybridisation (CGH) is a novel approach for the detection of cytogenetic abnormalities. It combines flow sorting of chromosomes, degenerate oligonucleotide primed (DOP)-PCR and a modified comparative genome hybridisation (CGH) technique to define the site and extent of intrachromosomal duplications. Chromosome specific paint probes for aberrant chromosomes and their normal homologues from four subjects with unbalanced duplications within chromosomes 2p11-15, 3q25-26, 5q34-qter, and 12q23-24.2 were made. They were then cohybridised on normal metaphase spreads and the ratio of their relative intensities of hybridisation analysed. The results were compared to those of similar experiments where regular CGH was performed on the same four patients. We provide evidence that this method can detect duplications and deficiencies which might be missed by conventional CGH, as the ratio of hybridisation of abnormal/normal DNA is 2:1 rather than 3:2. It is the method of choice where mosaicism is present or where only one of several homologous chromosomes is duplicated. Furthermore, it suggests that DOP-PCR amplifies all or most of the euchromatic regions of the genome equally.
The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a fast and economical means of assaying SNP's, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. To improve the throughput and efficiency of T-ARMS-PCR, we combined T-ARMS-PCR with a chimeric primer-based temperature switch PCR (TSP) strategy, and used capillary electrophoresis (CE) for amplicon separation and identification. We assessed this process in the simultaneous genotyping of four breast cancer–and two cervical cancer risk–related SNPs.
A total of 24 T-ARMS-PCR primers, each 5′-tagged with a universal sequence and a pair of universal primers, were pooled together to amplify the 12 target alleles of 6 SNPs in 186 control female blood samples. Direct sequencing of all samples was also performed to assess the accuracy of this method.
Of the 186 samples, as many as 11 amplicons can be produced in one single PCR and separated by CE. Genotyping results of the multiplex T-ARMS-PCR were in complete agreement with direct sequencing of all samples.
This novel multiplex T-ARMS-PCR method is the first reported method allowing one to genotype six SNPs in a single reaction with no post-PCR treatment other than electrophoresis. This method is reliable, fast, and easy to perform.
Background/Aim—The pathogenetic relation between liver cell dysplasia and hepatocellular carcinoma (HCC) is poorly understood. The aim of this study was to determine whether there is a genetic link between liver cell dysplasia and HCC that could support the role of dysplasia as a tumour precursor lesion.
Methods—Microdissection from paraffin wax embedded sections and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) were combined to analyse chromosomal imbalances by comparative genomic hybridisation (CGH) in nine HCCs and nodules containing liver cell dysplasia and cirrhosis adjacent to the tumours. Seven cases of large cell changes (LCC) and three cases of small cell changes (SCC) were analysed. The genetic abnormalities detected in liver cell dysplasia were then compared with those present in the corresponding HCC.
Results—No abnormalities were detected in LCC and cirrhotic nodules, arguing against the preneoplasic nature of these cell foci. In contrast, a subset of chromosomal alterations present in HCCs was found in the adjacent SCC.
Conclusions—These findings support the preneoplastic status of SCC in human hepatocarcinogenesis.
comparative genomic hybridisation; hepatocellular carcinoma; liver cell dysplasia
Whole genome amplification methods are a recently developed tool for amplifying DNA from limited template. We report its application in trypanosome infections, characterised by low parasitaemias. Multiple Displacement Amplification (MDA) amplifies DNA with a simple in vitro step, and was evaluated on mouse blood samples on FTA filter cards with known numbers of Trypanosoma brucei parasites. The data showed a twenty-fold increase in the number of PCRs possible per sample, using primers diagnostic for the multi-copy ribosomal ITS region or 177 bp repeats, and a twenty-fold increase in sensitivity over nested PCR against a single copy microsatellite. Using MDA for microsatellite genotyping caused allele dropout at low DNA concentrations, which was overcome by pooling multiple MDA reactions. The validity of using MDA was established with samples from Human African Trypanosomiasis patients. The use of MDA allows maximal use of finite DNA samples and may prove a valuable tool in studies where multiple reactions are necessary, such as population genetic analyses.
Amplification by polymerase chain reaction is often used in the preparation of template DNA molecules for next-generation sequencing. Amplification increases the number of available molecules for sequencing but changes the representation of the template molecules in the amplified product and introduces random errors. Such changes in representation hinder applications requiring accurate quantification of template molecules, such as allele calling or estimation of microbial diversity. We present a simple method to count the number of template molecules using degenerate bases and show that it improves genotyping accuracy and removes noise from PCR amplification. This method can be easily added to existing DNA library preparation techniques and can improve the accuracy of variant calling.
The objective of this study was to develop a real-time polymerase chain reaction (PCR) method to detect MDR1 (human multidrug resistance gene) single nucleotide polymorphisms (SNPs) C3435T and G2677T. C3435T and G2677T are linked to MDR1*2, which is associated with enhanced efflux activity in vitro. Using the Smart Cycler, an allele-specific real-time PCR-based genotyping method was developed to detect C3435T and G2677T. The MDR1 genotype of human genomic DNA templates was determined by direct DNA sequencing. PCR reactions for genotyping C3435T and G2677T by using allele-specific primers were conducted in separate tubes. An additional nucleotide mismatch at the third position from the 3′ end of each allele-specific primer was used to abrogate nonspecific PCR amplification. The fluorescence emitted by SYBR Green I was monitored to detect formation of specific PCRproducts. PCR growth curves exceeding the threshold cycle were considered positive. Fluorescence melt-curve analysis was used to corroborate results from PCR growth curves. Using PCR growth curves, our assay accurately determined hetero- and homozygosity for C3435T and G2677T. Genotype assignments based on PCR growth curve, melt-curve analysis, agarose gel electrophoresis, and direct DNA sequencing results of PCR products were in perfect agreement. We have developed a rapid MDR1 genotyping method that can be used to assess the contribution of MDR1*2 to pharmacokinetic and pharmacodynamic variability of P-glycoprotein substrates.
P-glycoprotein; single nucleotide polymorphism; real-time polymerase chain reaction