Evaluation of procedures for amplification of small-size samples for hybridization on microarrays

Various approaches have been developed for the preparation of samples for gene expression monitoring. For Affymetrix chips, a standard protocol is widely used; however, this is inefficient for small samples such as laser capture microdissections. Several amplification procedures for such samples already exist, and our goal was to test two of them: the first is based on random PCR amplification, and the second, linear amplification, involves performing the standard protocol twice. We analyzed a dilution of a commercially available mouse brain total RNA preparation and microdissections from mouse hippocampus and striatum. We evaluated the quality of microarray data by analyzing several chip parameters and performing multiple comparisons. At the biological level, brain microdissections prepared with either method gave similar expression results. At the technical level, analysis of the commercial sample showed that random PCR amplification is more reproducible, requires smaller RNA input, and generates cRNA of higher quality than linear amplification.     

Reliable amplification method for bacterial RNA

DNA microarray technology has been increasingly applied for studies of clinical samples. Frequently, RNA probes from clinical samples are available in limited amounts. We describe a reliable amplification method for bacterial RNA. We verified this method on mycobacterial RNA applying mycobacterial genome-directed primers (mtGDPs). Glass slide-based oligoarrays were employed to assess the quality of the amplification method. We observed a relatively small bias in amplified RNA pool when compared to the unamplified one. Up to 1000-fold linear RNA amplification in a single amplification round was obtained. To our knowledge, this study describes the first amplification method for mycobacterial RNA.     

Use of commercial cards for freeing DNA from mycobacterial strains

A new commercial system for rapid extraction of DNA, consisting of a card on which a drop of bacterial suspension is spotted, was evaluated with 43 mycobacterial strains. Once dried, a small disk of the seeded area was directly transferred in the amplification mixture. All the samples produced good quality amplification products which were satisfactorily sequenced.     

SNP-based chromosomal copy number ascertainment following multiple displacement whole-genome amplification

Whole genome amplification by multiple displacement amplification (MDA) offers investigators using precious genomic DNA samples a high fidelity method for amplifying nanogram quantities of DNA several thousandfold. This becomes especially important for the modemrn day genomics researcher who more and more commonly is applying today's genome scanning technologies to patient cohort samples collected years ago that are irrecoverable and invariably in short supply. We present evidence here that MDA-prepared genomic DNA includes artifacts of chromosomal copy number that resemble copy number polymorphisms (CNPs) upon analysis of the DNA on the Affymetrix 10K GeneChip. The study of CNPs in both health and disease is a rapidly growing area of research, however our current understanding of the relevance of CNPs is incomplete. Our data indicate that utilization of whole genome-amplified samples for analysis heavily reliant on accurate copy number retention could be confounded if the genomic DNA sample was subjected to MDA. We recommend that small amounts of patient cohort DNA stocks be set aside and not subjected to whole genome amplification in order to facilitate the unbiased determination of chromosomal copy numbers when desired.     

降香黄檀的DNA条形码鉴定研究

DNA条形码技术是利用基因组中一段短的标准序列进行物种的鉴定并探索其亲缘进化关系。本研究对采自海南不同地区降香黄檀五个居群24份样品的psbA-trnH,rbcL,核ITS及ITS2序列进行PCR扩增和测序,比较各序列扩增和测序效率。种间和种内变异,采用BLAST1和邻接 (NJ) 法构建系统聚类树方法评价不同序列的鉴定能力。结果表明ITS2在所研究的材料中具有最高的扩增和测序效率,而ITS扩增效率较低。ITS2完整序列在区分黄檀属不同种间差异具有较大优势。因此可利用ITS2从分子水平区分降香黄檀与其他混伪种。     

Assessment of amplicons in the DNA from boiled tissue by PCR and AP-PCR amplification

Polymerase chain reaction (PCR) is a technique sensitive enough to amplify small DNA fragments a billion-fold. The generation of amplicons either by PCR with a set of oligo primers or by arbitrarily primed AP-PCR with a single oligonucleotide primer is based on the availability of intact template and priming sites. With these approaches, it is possible to generate specific and random amplicons to assess the extent of damage to DNA caused by any of the physical, chemical, or environmental factors. We report the amplification of sex chromosome and autosome specific loci in the buffalo (Bubalus bubalis) genome by symmetrical and AP-PCR performed on DNA samples isolated from the muscle tissues that were boiled (treated) for different lengths of time. No difference was noticed in the amplification profile of DNA cooked for various lengths 0f time. However, after HinfI treatment, AP-PCR amplification of these DNAs revealed more bands on agarose gel than unrestricted samples. The successful amplification of the DNA samples isolated from the boiled tissues is attributed to the intactness of the amplicons. This suggests that despite storage for more than a year and subsequent heat treatment to the muscle tissues, the DNA remains a good substrate for PCR and AP-PCR amplification. Relevance of this work in the context of DNA probe technology is discussed.     

Immobilized Sample Amplification for Quantitative Determination of Retroviruses

Immobilized sample amplification (ISA) is a novel method for amplification, detection, monitoring, and quantitative determination of nucleic acids from a minute amount of sample. We present here a novel quantitative ISA assay for retroviruses using a replication-defective recombinant retrovirus as a model retrovirus. Samples, as small as 5 to 10 microl or as large as 1 ml or more in volume, are readily immobilized on a nylon or polyester matrix. Retroviral RNA is directly amplified following the rehydration of the immobilized samples, thus eliminating the needs for retroviral RNA extraction. An ISA assay of a 10-microl viral sample generates results equal to or better than that of RT-PCR on equivalent amount RNA isolated from larger sample volumes. Recovery of RNA from small volumes, such as 10 microl, is almost impossible, whereas ISA assay detects retroviruses from as small as 1 to 5 microl of viral samples containing 10(4) cfu/ml determined by colony-forming assay. Extraction of RNA from a small amount of infectious viral samples not only is a difficult, biohazardous procedure, but also introduces random errors which contribute to variability in viral quantitation. Since the ISA method eliminates the isolation/extraction of the nucleic acids, it significantly shortens the handling time for the biohazardous materials and simplifies the procedure for analyzing small quantities of biological samples. This method detects less than 10 infectious retroviral particles as determined by both colony-forming assay and electron microscope studies. The format and protocol of this quantitative ISA assay can be easily automated to fit into numerous platforms, thus making it attractive for laboratory automation.     

PCR amplification of streptococcal DNA using crude cell lysates

Gram-positive organisms such as streptococci and enterococci are often difficult to lyse. Obtaining DNA for procedures such as PCR amplification usually requires a large scale isolation for each strain under investigation. We describe a simple procedure for small volumes of whole cells, involving pretreatment with detergent and proteinase that allows for efficient release of DNA for PCR amplification. This procedure is fast, reproducible, can be used with a large number of samples, and has been successfully applied to a variety of streptococcal and enterococcal strains.     

Evaluation of methods for single hair DNA amplification

Because of the low amount of DNA in single hairs, it may be difficult to obtain reliable genotypes for forensic and conservation genetics studies. We therefore compared different methods for reliably genotyping single hair samples. Our results indicate that preliminary whole genome amplification can increase the likelihood of successfully genotyping a single hair compared to other commonly used protocols. The difference between the methods is small for single locus comparisons, but it becomes more important in multi-locus comparisons. The economic and time costs of the whole genome amplification may prevent its large-scale use in non-invasive monitoring programs. Nevertheless, it may be a very useful approach for the analysis of especially valuable samples.     

Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments

Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and phi 29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations >or= 10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples.     

A universal primer set for PCR amplification of nuclear histone H4 genes from all animal species

To control the quality of genomic DNA of samples from a wide variety of animals, a heminested PCR assay specifically targeting a nuclear gene has been developed. The histone H4 gene family comprises a small number of genes considered among the most conserved genes in living organisms. Tissue samples from necropsies and from cells belonging to 43 different species were studied, eight samples from invertebrates and 35 samples from vertebrates covering all classes. Ancient DNA samples from three Siberian woolly mammoths (Mammuthus primigenius) dating between 40,000 and 49,000 years before present were also tested for PCR amplification. Performance of HIST2H4 amplification were also compared with those of previously published universal PCRs (28S rRNA, 18S rRNA, and cytochrome b). Overall, 95% of species studied yielded an amplification product, including some old samples from gorilla and chimpanzees. The data indicate that the HIST2H4 amplimers are, thus, suitable for both DNA quality testing as well as species identification in the animal kingdom.     

Molecular genetic analysis of the polymorphic apolipoprotein E in modern and ancient human DNA samples

In this report, methodical bases for the molecular genetic analysis of the three common apolipoprotein E alleles APOE*2, APOE*3 and APOE*4 in DNA isolated from ancient human skeletal remains are described. Considering that ancient DNA target regions for amplification are generally quite small, the detection method is based on short amplification products in the range from 71 bp to 75 bp. The applicability of the modified method for APOE genotyping was examined in modern human DNA samples.     

Automated sequencing of complete mitochondrial genomes from laser-capture microdissected samples

Mitochondrial DNA mutations have been related to both aging and a variety of diseases such as cancer. Due to the relatively small size of the genome (16 kb) and with the use of automated DNA sequencing, the entire genome can be sequenced from clinical specimens in days. We present a reliable approach to complete mitochondrial genome sequencing from laser-capture microdissected human clinical cancer specimens that overcome the inherent limitations of relatively small tissue samples and partial DNA degradation, which are unavoidable when laser-capture microdissection is used to attain pure populations of cells from heterogeneous tissues obtained from surgical procedures. The acquisition of sufficient template combined with a standard set of 18 pairs of PCR primers allows for the efficient amplification of the genome. Subsequent single-stranded amplification is performed using 36 sequencing primers, and samples are run on an ABI PRISM 3100 Genetic Analyzer. The use of this procedure should allow even investigators with little experience sequencing from clinical specimens success in complete mitochondrial genome sequencing.     

A primer set to determine sex in the small Indian mongoose, Herpestes auropunctatus

To enable the accurate sexing of individuals of introduced populations of the small Indian mongoose, Herpestes auropunctatus, we designed a primer set for the amplification of the sex-specific fragments EIF2S3Y and EIF2S3X. Using this primer set, the expected amplification products were obtained for all samples of genomic DNA tested: males yielded two bands and females a single band. Sequencing of each PCR product confirmed that the 769-bp fragment amplified from DNA samples of both sexes was derived from EIF2S3X, whereas the 546-bp fragment amplified only from male DNA samples was derived from EIF2S3Y. The results indicated that this primer set is useful for sex identification in this species.     

Real time PCR profiling of 330 human micro-RNAs

The small size of miRNAs makes profiling of all the 462 known human miRNAs difficult using single cell samples. Recently, we showed that judicious sequence partitioning between RT primers and second strand synthesis primers permitted multiplexed RT-PCR amplification of miRNA in very small samples to allow individual real time PCR quantification. Here, we show that zip coding the primers and TaqMan probes with sequences specific to each miRNA greatly improves reaction specificity, which permits the profiling of all miRNAs in a single multiplexed RT-PCR reaction.     

Impact of whole genome amplification on analysis of copy number variants

Large-scale copy number variants (CNVs) have recently been recognized to play a role in human genome variation and disease. Approaches for analysis of CNVs in small samples such as microdissected tissues can be confounded by limited amounts of material. To facilitate analyses of such samples, whole genome amplification (WGA) techniques were developed. In this study, we explored the impact of Phi29 multiple-strand displacement amplification on detection of CNVs using oligonucleotide arrays. We extracted DNA from fresh frozen lymph node samples and used this for amplification and analysis on the Affymetrix Mapping 500k SNP array platform. We demonstrated that the WGA procedure introduces hundreds of potentially confounding CNV artifacts that can obscure detection of bona fide variants. Our analysis indicates that many artifacts are reproducible, and may correlate with proximity to chromosome ends and GC content. Pair-wise comparison of amplified products considerably reduced the number of apparent artifacts and partially restored the ability to detect real CNVs. Our results suggest WGA material may be appropriate for copy number analysis when amplified samples are compared to similarly amplified samples and that only the CNVs with the greatest significance values detected by such comparisons are likely to be representative of the unamplified samples.