Primer Extension Preamplification Research Articles

Effectiveness of whole genome amplification prior to short tandem repeat analysis for degraded DNA

Short tandem repeat (STR) analysis is prone to failure as DNA is frequently damaged by various environmental factors; hence, increasing the number of starting templates may constitute a feasible approach to improve STR profiling success. Whole genome amplification (WGA) is often applied to bolster starting template quantity. Moreover, WGA can reportedly be used on degraded DNA samples in forensics. Therefore, we utilized a PCR-based WGA method, termed “modified improved primer extension preamplification” (mIPEP), prior to STR analysis of degraded DNA, as this method is less affected by DNA quantity and quality than most others. Saliva from four volunteers was dried on glass fiber filter papers (paper) and glass slides (glass) and irradiated with UVA light (365 nm). The mIPEP method was initiated using 5, 0.5, and 0.05 ng of DNA following DNA extraction. The DNA degradation index (DI) was calculated based on the ratio of 129 to 41 bp DNA fragments; lower numbers indicate higher degradation. Following mIPEP, STR analysis was performed using the AmpFlSTR Identifiler PCR amplification kit. The number of detectable STR loci, with and without mIPEP, decreased according to reduced DI in a different manner for the various DNA concentrations extracted from paper and glass. Specifically, for the 5 ng DNA sample on paper, at a DI < 0.2, the number of detectable STR loci was greater with mIPEP than without it, owing to fewer locus drop-outs. Similarly, the 0.05 ng DNA sample deposited on paper, at DI ≥ 0.7, exhibited higher numbers of detectable STR loci when prepared using mIPEP owing to fewer allele drop-outs. Moreover, among samples deposited on glass, the 0.05 ng DNA sample at DI ≥ 0.4 afforded a larger number of detectable STR loci when prepared using mIPEP than those without mIPEP, owing to fewer locus drop-outs. These findings suggest that performing mIPEP in accordance with sample DNA condition (e.g., quantity and quality) may lead to increased success of STR analysis. Notably, the conditions identified as most responsive to mIPEP were consistent across both UVA-irradiated and environmentally-damaged sample states. Taken together, our results suggest that applying mIPEP would be beneficial to obtain improved STR profiles under conditions involving severely degraded samples with large quantities of DNA, or with small quantities of DNA albeit with slight degradation.

Investigation of the efficiency of whole genome amplification prior to short tandem repeat analysis using degraded DNA

Short tandem repeat (STR) analysis is prone to failure because DNA is frequently degraded by various environmental factors. Increasing the number of starting templates may improve success in STR profiling. One approach to increase the number of DNA templates is whole genome amplification (WGA); however, few studies have demonstrated that WGA can be used on degraded DNA samples in forensics. Therefore, we performed a PCR-based WGA termed “modified improved primer extension preamplification” (mIPEP) prior to STR analysis of degraded DNA, since this method was less affected by DNA quantity and quality than other approaches are.Saliva from four volunteers was dried onto filter papers. These samples were irradiated with UVA light (365 nm) for 3, 7, 14, and 30 days. The mIPEP method was initiated using 5, 0.5, and 0.05 ng of DNA after DNA extraction. Following mIPEP, STR analysis was performed using the AmpFlSTR Identifiler PCR Amplification Kit. This study was approved by the ethics committee at Tokyo Women’s Medical University.The number of detectable STR loci in 0.05 ng DNA sample was greater in those prepared by mIPEP than those prepared without mIPEP, when no radiation was applied. After 3 days of irradiation, the number of detectable STR loci in 0.5 ng DNA sample was smaller in those treated with mIPEP than in samples without mIPEP. There was no significant difference between the number of detectable STR loci with and without mIPEP after 7 and 14 days of irradiation. However, after 30 days of irradiation, the number of detectable STR loci in 5 ng DNA samples prepared with mIPEP was greater than in those without mIPEP. Hence, mIPEP was not necessary for STR loci detection in moderately degraded samples, but it did improve the success of STR profiling in severely degraded samples.

Genotyping of Individually Isolated gametes from Gossypium hirsutum Chromosome Substitution Lines

As an important crop for US agricultural security and sustainable agriculture, cotton genetic improvement requires development of innovative analytical tools. Genetic analyses based on individual gametes provide several advantages including minimum sample requirement, genetic identification of male parent and overcoming the complexity of polyploidy due to haploid nature of microspores. This research used tetraploid cotton (G. hirsutum x G. barbadense) chromosome substitution (CS) lines with G. barbadense chromosomes 17 and 25 in G. hirsutum background. The individually isolated pollen grains were germinated to release their DNAs and genomic DNA was increased by modified primer extension pre-amplification (PEP) using MasterAmp™ Extra-Long PCR Kit (EPICENTRE®, Madison, WI). Also from select cotton lines, microspores just released from tetrad developmental stage were individually isolated and gamete DNA was extracted as well as amplified through multiple displacement amplification (MDA) using REPLI-g Single Cell Kit (QIAGEN, Valencia, CA). The parental samples along with PEP and MDA amplified individual gamete DNAs were then analyzed using simple sequence repeat (SSR) as well as IRD800 and IRD-700 labeled (Li-Cor, Lincoln, NE) amplified fragment length polymorphism (AFLP) methods. Nineteen SSR and 28 AFLP primer pairs were used to analyze TM-1, 3-79, CS-B17 and CS-B25 cotton lines. The amplification of parental SSR and AFLP markers from both mature pollen as well as early free microspore samples provided unique tools for comparative genetic studies using agarose and polyacrylamide gel electrophoresis, respectively.

The influence of high temperature on the possibility of DNA typing in various human tissues.

The identification of unknown victims of high temperatures (fire, terrorist attack, and other disasters) is one of the most difficult tasks faced by forensic geneticists. The main aim of this study was to in­vestigate the availability of DNA isolated from various human tissue samples exposed to high temperatures of 100–1000°C for 5 and 10 minutes. Samples of varying thickness of thigh muscle, liver, heart, adipose tissue, bone, teeth, hair and nails of 52 fresh cadavers and 59 healthy teeth of 29 volunteers were used. The study was performed using the following commercially available STR (Short Tandem Repeats) and miniSTR kits: AmpFlSTR®SGM Plus® and AmpFlSTR®MiniFilerTM. Hyper variable region I (HVI) of human mitochondrial DNA (mtDNA) was sequenced with BigDye Terminator Cycle Sequencing Kit 1.1. The PEP (Primer-Extension Preamplification) method was used for the whole human genome amplification. It was possible to obtain complete DNA profiles (AmpFlSTR®SGM Plus®, AmpFlSTR®MiniFilerTM Applied Biosystems, USA and mtDNA HVI region) for tissue samples of heart, liver and thigh muscle, exposed up to 900°C for 5 min. However, under the applied conditions, limited usefulness of hair, nails and teeth for identification purposes was shown. DNA stability in tissues subjected to incineration depends on many factors, like tissue type and its thickness, temperature and time of exposure. In the cases of human remains exposed to high temperatures, samples of soft tissues of the highest weight (thickness) provide the best chance of successful identification through the genetic analysis. In some cases of negative results, even if using mtDNA typing, application of the whole genome amplification (WGA) technique could provide the expected results for highly degraded DNA templates.

High-throughput genotyping of Anopheles mosquitoes using intact legs by Agena Biosciences iPLEX.

Recent developments in genotyping technologies coupled with the growing desire to characterize genome variation in Anopheles populations open the opportunity to develop more effective genotyping strategies for high-throughput screening. A major bottleneck of this goal is nucleic acid extraction. Here, we examined the feasibility of using intact portions of a mosquito's leg as sources of template DNA for whole-genome amplification (WGA) by primer-extension preamplification. We used the Agena Biosciences MassARRAY(®) platform (formerly Sequenom) to genotype 78 SNPs for 265 WGA leg samples. We performed nucleic acid extraction on 36 mosquito carcasses and compared the genotype call concordance with their corresponding legs and observed full concordance. Using three legs instead of one improved genotyping success rates (96% vs. 89%, respectively), although this difference was not significant. We provide a proof of concept that WGA reactions can be performed directly on mosquito legs, thereby eliminating the need to extract nucleic acid. This approach is straightforward and sensitive and allows both species determination and genotyping of Anopheles mosquitoes to be performed in a high-throughput manner. Our protocol also leaves the mosquito body intact facilitating other experimental analysis to be undertaken on the same sample. Based on our findings, this method would also be suitable for use with other insect species.

Isolation and DNA-Marker Based Genotyping of Individual Pollen Grains

Dry pollen of diploid Solanum clone 9507-04, Cotton chromosomal substitution line and Echinacea purpurea „Magnus‟ were isolated and their cells contents released in individual tubes containing water or pollen germination media, respectively.Six polymorphic markers from individual pollen grains of the diploid potato were amplified using two random amplified polymorphic DNA (RAPD) primers, i.e., UBC291 and UBC504. The procedures for primer extension pre-amplification (PEP) for genome and subsequent amplified fragment length polymorphism (AFLP) analysis with 13 primers pairs were tested on the upland cotton line. The cotton parental AFLP markers were scored for their presence or absences in the 35 pollen samples and majority of the markers were found monomorphic as expected from the gametes of a near isogenic line.PEP and AFLP procedures for linkage mapping were standardized usingpurpleconeflower pollen grains. From 64 possible AFLP primer pairs,37.5% amplified 104 markers for coneflowerthat were linked in 11 linkage groupsranging from 13 to 134 centimorgans (cM). The 409 cM sizedlinkage map constructed forE. purpurea „Magnus‟genome had average distance of 2.4 cM to 22 cMfor the two to 55 polymorphic markers identifiedwithin the 11 groups.The protocols described in this report would serve to enhance breeding efficiencies in plantsimportant for food,fiber, ornamental and medicinal industries.

Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR

PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists.

Different whole-genome amplification methods as a preamplification tool in Y-chromosome Loci analysis.

Degraded and low template DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded and low template DNA is of great importance, because its results impact the quality and reliability of expert testimonies. Recently, a number of whole-genome amplification (WGA) methods have been proposed as preamplification tools improving quantity and quality of DNA. We chose, investigated, and compared 7 WGA methods to evaluate their ability to "recover" degraded and nondegraded DNA. These methods include degenerate oligonucleotide primed polymerase chain reaction, primer extension preamplification (PEP) polymerase chain reaction, GenomePlex WGA (Sigma), multiple displacement amplification, GenomiPhi Amplification Kit (Amersham Biosciences), restriction and circularization aided rolling circle amplification, and blunt-end ligation-mediated WGA. Recently, we have described the comparison of these methods' efficiency and reliability using SGMPlus kit. However, Y-chromosome profiling is also often used in analysis of both nondegraded and degraded DNA. This includes criminal cases and investigation of kinship in male linage. Here we demonstrate the impact of WGA methods on Y-chromosome loci (Yfiler) reactivation.The best results for nondegraded DNA were obtained with GenomiPhi kit and PEP method. In the case of degraded DNA (200 base pairs), the most complete profiles were obtained with GenomePlex kit and PEP method. None of the analyzed methods allowed full reactivation of degraded (200 base pairs) DNA in terms of Y-chromosome loci profiling.

Whole genome amplification of degraded and nondegraded DNA for forensic purposes

Degraded DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded DNA is of great importance, since its results impact the quality and reliability of expert testimonies. Recently, a number of whole genome amplification (WGA) methods have been proposed as preamplification tools. They work on the premise of being able to generate microgram quantities of DNA from as little as the quantity of DNA from a single cell. We chose, investigated, and compared seven WGA methods to evaluate their ability to “recover” degraded and nondegraded DNA: degenerate oligonucleotide-primed PCR, primer extension preamplification PCR, GenomePlex™ WGA commercial kit (Sigma), multiple displacement amplification, GenomiPhi™ Amplification kit (Amersham Biosciences), restriction and circularization-aided rolling circle amplification, and blunt-end ligation-mediated WGA. The efficiency and reliability of those methods were analyzed and compared using SGMPlus, YFiler, mtDNA, and Y-chromosome SNP typing. The best results for nondegraded DNA were obtained with GenomiPhi and PEP methods. In the case of degraded DNA (200 bp), the best results were obtained with GenomePlex which successfully amplified also severely degraded DNA (100 bp), thus enabling correct typing of mtDNA and Y-SNP loci. WGA may be very useful in analysis of low copy number DNA or degraded DNA in forensic genetics, especially after introduction of some improvements (sample pooling and replicate DNA typing).Electronic supplementary materialThe online version of this article (doi:10.1007/s00414-012-0764-9) contains supplementary material, which is available to authorized users.

Evaluating the performance of whole genome amplification for use in low template DNA typing

We report on the performance of two whole genome amplification methods, GenomiPhi™ amplification and modified-improved primer extension preamplification (mIPEP), when analysing low template DNA samples. Template as low as 10 pg treated with mIPEP generated more than 1 ng of DNA that could be used in STR typing. Initial templates of 100-10 pg, when treated with mIPEP, generated an increase in alleles compared with control samples. Partial profiles using the AmpFℓSTR(®) Identifiler™ Kit were produced from this suboptimal DNA template, with 70% of the possible alleles (21.7 ± 2.1 in 32 alleles) recorded, using the mIPEP amplified products with an initial template of 100 pg. Allelic imbalance decreased with samples treated with whole genome amplification method (WGA) compared with those without this initial treatment. Further methods for improvement were also analysed including altering the condition of electrokinetic injection, and the successful DNA typing rate was increased to about 80%. This report illustrates the potential use and limitations of WGA for low template samples.

A Short and Simple Improved-Primer Extension Preamplification (I-PEP) Procedure for Whole Genome Amplification (WGA) of Bovine Cells

Embryo transfer is a reproductive technique that has a major impact on the dissemination of economically important genes and the rate of genetic gain in breeding schemes. In recent years, there has been increasing interest in the use of sexed and genotyped embryos in commercial embryo transfer programs. Marker/gene assisted selection (MAS / GAS) projects can be performed in the pre-implantation stage through mass production of characterized embryos. Biopsy of a few cells in the morulla stage is essential for pre-implantation genetic diagnosis (PGD), in which sex determination, evaluation of disease genes, and genotyping for candidate genes are performed. Limited quantity of cells and low amount of DNA restrict the use of multiple molecular analyses in PGD programs. Recently, whole genome amplification (WGA) techniques promise to overcome this problem by providing sufficient input DNA for analysis. Among several techniques proposed for WGA, the primer extension pre-amplification (PEP) and the improved-primer extension pre-amplification (I-PEP) methods are the most commonly used. However, these methods are time-consuming and need more than 12 h amplification cycles. Since the time is a critical parameter in the successful characterized embryo transfer, the shortening of diagnosis time is highly desirable. In this study, we developed a short and simple I-PEP procedure (∼3 h) and evaluated its performance for the amplification of bovine genomic DNA. We assessed short WGA procedure by polymerase chain reaction (PCR) amplification of 7 specific loci. The results indicated that the short procedure possesses enough sensitivity for the molecular genetic analysis of 1 input cell. Although the efficiency of the method was 100%, there was an inconsistency between genomic DNA (gDNA) and whole genome amplification product (wgaDNA) genotypes for kappa-casein locus; that is, however, most likely due to allele drop-out (ADO) or false homozigocity. The results of this study indicate that with the application of reliable methods, WGA-amplified bovine DNA will be a useful source for sexing and genotyping bovine embryos in several quantitative trait locus (QTL) markers.

Chromosome copy analysis by single-cell comparative genomic hybridization technique based on primer extension preamplification and degenerate oligonucleotide primed-PCR

To establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD). Twelve single-cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR) amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed. The amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR, and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods. PEP-DOP-PCR can effectively amplify the whole genome DNA of single cell. Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneously, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.

Single‐Cell DNA and FISH Analysis for Application to Preimplantation Genetic Diagnosis

Preimplantation genetic testing, which includes preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS), is a form of a very early prenatal testing. The goal of this method is to avoid transfer of embryos affected with a specific genetic disease or condition. This unit describes the steps involved in amplifying DNA from a single blastomere and specific assays for detecting a variety of DNA mutations. For some assays, whole-genome amplification by primer-extension preamplification (PEP) is performed prior to analysis. Support protocols describe the biopsy of one or two blastomeres from the developing preimplantation embryo, isolation for further investigation of all blastomeres from embryos shown to have the mutant allele, and isolation of single lymphocytes or lymphoblastoid cells as models for single-cell DNA analysis. A procedure for FISH analysis on single interphase blastomeres is provided along with support protocols for probe preparation and probe validation, which is recommended as a preliminary step before performing any PGD or PGS FISH analysis.

Specific and complete human genome amplification with improved yield achieved by phi29 DNA polymerase and a novel primer at elevated temperature

BackgroundsWhole 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.FindingsEffects 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.ConclusionThe use of new primers in this study combined with elevated incubation temperatures in MDA has remarkably improved the specificity, amplification yield, and suppressed TIDA.

Quantitative abnormalities of fetal trophoblast cells in maternal circulation in preeclampsia

To determine if quantitative abnormalities of circulating fetal trophoblast cells (CFTCs) are associated with preeclampsia. The trophoblast cell-specific antibody, MEM-G/9 (monoclonal antibody to HLA-G), was applied to distinguish the trophoblast cells from the maternal circulation. The trophoblast cells were isolated by density-gradient centrifugation from maternal blood samples of normal pregnant and preeclamptic women, respectively. After preliminary enrichment, the CFTCs were identified by immunocytochemical staining with the MEM-G/9. To prove fetal origin of the HLA-G-positive cells, primer-extension preamplification (PEP) and polymerase chain reaction (PCR) based on single HLA-G-positive cells were adopted to detect human sex-determining region of the Y-chromosome (SRY) gene. There were 6.88 +/- 1.54 and 30.56 +/- 5.16 HLA-G-positive cells in 6 mL maternal blood from the normal pregnant (n = 16) and preeclamptic women (n = 18), respectively. The difference was statistically significant (p < 0.001). The SRY gene from the HLA-G-positive cells was detected in all pregnant women carrying male fetuses. The sensitivity and specificity of PEP and PCR for the SRY gene detection were 100%. It is concluded that enhancement of CFTCs numbers is related to preeclampsia, which provides information useful for noninvasive prenatal diagnosis of preeclampsia.

Enrichment of circulating fetal nucleated red blood cell for non-invasive prenatal diagnosis with a new polyclonal antibody specific to fetal hemoglobin

Objective To investigate the feasibility of a new polyclonal antibody specific to fetal hemoglobin (HbF) and its application in enrichment of circulating fetal nucleated red blood cell(NRBC) for non-invasive prenatal diagnosis. Methods A polyclonal antibody against a synthetic peptide comprising residues 69-78 of the γ-chain of HbF was prepared and conjugated to carrier protein KLH as the immunogen according to the specific antigenic determinant. The peptide-KLH solution was mixed with freund's complete or incomplete adjuvant and immunized goat to prepare specific polyclonal antibody against the γ-chain of fetal hemoglobin. After purification with protein G, maternal blood was obtained from 32 pregnant women at 22 to 39 weeks of gestation. NRBCs were separated and then stained with antibody against the γ chain of HbF. All the positive cells were collected by micromanipulator under microscopic observation, and whole genome was amplified by improved primer extension preamplification (PEP). Multiplex polymerase chain reaction amplification at nine different polymorphic short tandem repeat (STR) loci was also used to determine origin of the positive cells isolated from maternal blood. Results NRBCs stained with antibody against the γ chain of HbF were found in all of the blood from the 32 cases. Attached positive cells with anti-HbF staining have unique morphological characteristics, low nucleus-to-cytoplasm ratio, brown cytoplasm and blue dense nucleus after hematoxylin counterstain under microscopic observation, which can distinguished NRBCs with other cells. A total of 183 NRBCs were found in all of 32 pregnant women at a range of 0.6～1.8 cell/ml venous blood. The accurate rate was 90.6% by the STR genotype identification. Conclusion The antibodies specific to fetal γ-chain of fetal hemoglobin with synthetic peptide technology may have wide clinical utility in identification of fetal NRBCs from maternal circulation for non-invasive prenatal genetic diagnosis. Key words: Fetal hemoglobin; Erythroblasts; Prenatal diagnosis; Peptide biosynthesis

Methodology matters – prognostic significance of HER2 protein expression versus HER2 gene amplification in metastatic breast cancer

Whole genome amplification (WGA) has emerged as a fundamental method for DNA analysis from limited quantities of genomic DNA in forensic analysis and disease gene discovery. Several strategies for WGA have been developed during the past decade, each with variable fidelity, yield and coverage of the amplified genome. In the search for a reliable and robust WGA method for genotyping short tandem repeat (STR) loci and single nucleotide polymorphic (SNP) markers, we initially tested four common methods, viz., degenerate oligonucleotide primed PCR (DOP), primer extension preamplification (PEP), improved-PEP (I-PEP) and multiple displacement amplification (MDA), typing the 13 CODIS tetranucleotide repeat loci. The results showed among all methods, I-PEP and MDA have higher genomic coverage. DOP and PEP produced locus and allelic dropouts. Therefore, we emphasized on the evaluation of I-PEP and MDA protocols, which shows that the two methods have their relative strengths and weaknesses. In general, the product yield of MDA is higher than that of I-PEP. However, the specificity of the I-PEP products appears to be higher, particularly in the analysis of STR loci. In the analysis of SNP markers, some loci amplify better using products obtained from I-PEP whereas some worked better with MDA. Our analyses also demonstrate that blood spots on FTA cards are a more efficient source of DNA for I-PEP as compared to MDA, especially for STR analysis.

An efficient novel method for analyzing STR loci from a single sperm captured by laser microdissection

The identification of individuals using short tandem repeat (STR) analysis is important in forensic investigation. STR analysis is especially difficult using DNA from a single nucleus. In this study, DNA from a single sperm from a semen smear stained with hematoxylin and eosin (H&E) was captured using a laser microdissection method and amplified using the improved primer extension preamplification (I-PEP) polymerase chain reaction (PCR) method. Amplified DNA was used for STR analysis with the original and 3% primer methods. DNA from 15 STR loci and the amelogenin locus was amplified using an AmpFlSTR ® PCR Amplification kit (Applied Biosystems). Using the original primer method, it was impossible to genotype the 15 STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA). While using less primer (3% dilution), the 15 STR loci and amelogenin locus were determined perfectly in all of the preserved single sperms. Therefore, the reduced primer method is more efficient for analyzing STR loci from a single sperm captured by laser microdissection.

Whole-Genome Amplification by Improved Primer Extension Preamplification PCR (I-PEP-PCR)

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). Primer extension preamplification PCR (PEP-PCR), in contrast to degenerate oligonucleotide primed PCR (DOP-PCR), uses totally degenerate 15-mer PCR primers. An additional difference is that in PEP-PCR, the number of potential priming sites is orders of magnitude larger. The effectiveness of PEP-PCR has been increased by several alterations. The improved PEP (I-PEP) PCR approach, described in this protocol, uses a DNA polymerase cocktail that includes Taq DNA polymerase (to carry out the primer extension as in a traditional PCR) and a proofreading DNA polymerase (to provide 3'-to-5'-exonuclease activity, excising misincorporated nucleotides that slow the progression of Taq DNA polymerase). The result is far more efficient WGA, with increased fidelity due to the removal of the misincorporated nucleotides. Similar to DOP-PCR, PEP-PCR generates a smear of DNA fragments that are visible on an agarose gel.

Multiple Molecular Analyses From Minimal Cell Quantities by Sequential Isolation and Preamplification of DNA and RNA

Molecular analyses of single or very few cells are often handicapped by low amounts of DNA or RNA from starting material. Specimens like biopsies, embryonic stem cells, or laser-microdissected tissues often do not provide nucleic acid quantities required for robust amplification and verification by diagnostic polymerase chain reaction (PCR)-based analyses. A simultaneous isolation procedure for both RNA and DNA from a single sample would greatly facilitate demanding molecular studies. Here, we describe a new method that combines the isolation of mRNA using commercially available oligo (dT)-bound magnetic beads with the precipitation of DNA from the remaining cell lysate. Both types of nucleic acids can then be separately preamplified by improved primer extension preamplification and subsequently used for multiple molecular analyses. With this procedure we were able to reliably gain DNA for PCR from as few as 5 cells. RNA suitable for various reverse transcription-PCR approaches and sequencing analyses could simultaneously be isolated even from single cell preparations.