Increasing the denaturation temperature during the first cycles of amplification reduced allele dropout from single cells for preimplantation genetic diagnosis

Single cell polymerase chain reaction (PCR) for preimplantationgenetic diagnosis (PGD) needs to be highly efficient and accurate.In some single cells from human embryos presumed to be heterozygousfor the     

Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR: implications for preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells (polar bodies or blastomeres). Despite the use of increasingly robust protocols, allele drop-out (ADO; the failure to amplify one of the two alleles in a heterozygous cell) remains a significant problem for diagnosis using single cell PCR. In extreme cases ADO can affect >40% of amplifications and has already caused several PGD misdiagnoses. We suggest that an improved understanding of the origins of ADO will allow development of more reliable PCR assays. In this study we carefully varied reaction conditions in >3000 single cell amplifications, allowing factors influencing ADO rates to be identified. ADO was found to be affected by amplicon size, amount of DNA degradation, freezing and thawing, the PCR programme, and the number of cells simultaneously amplified. Factors found to have little or no affect on ADO were local DNA sequence, denaturing temperature (94 or 96 degrees C) and cell type. Consideration of the causal factors identified during this study should permit the design of PGD protocols that experience little ADO, thus improving the accuracy of PGD for single gene disorders.     

单管双向等位基因专一性扩增的单核苷酸多态分型的新方法

目的：建立一种基于等位基因专一性PCR原理的单核苷酸多态（single nucleotidepolymorphism,SNP)分型新方法：单管双向等位基因专一性扩增（single-tube bi-directional allele specificamplification,SB－ASA）,并考察专一性引物的3’端第3位碱基不配对对特异延伸的影响。方法：一个PCR反应体系包含两个3’末端分别与SNP两个等位基因特异结合的引物,它们延伸方向相反,产生长度不同的等位基因专一扩增产物,同时在两个等位基因特异性引物的3’端第3位碱基引入不配对以增加特异性。PCR产物经琼脂糖凝胶电泳后分析确定样本的基因型。观察在不同的温度条件下,近3’末端引入与不引入碱基不配对时两种引物特异延伸的情况,比较两种引物能特异延伸的退火温度（annealingtemperature,Ta)范围。结果：对于4个不同类型的SNP位点,SB－ASA都成功地分型了36个样本,与直接测序的结果完全一致。两条专一性引物3’物第3位碱基引入不配对后,能特异延伸的退火温度Ta范围分别从64℃～69℃、60℃～62℃扩大到46℃～66℃、56℃～61℃。结论：SB－ASA是一种简单快速而有效的SNP分型新方法;在等位基因专一性PCR体系中,专一性引物3’端第3位碱基引入不配对能增加引物对两个等位基因的区分能力。     

Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases

Molecular Human Reproduction, Vol. 9,     

Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases

To perform preimplantation genetic diagnosis for women carrying heteroplasmic mitochondrial DNA (mtDNA) mutations, it is necessary to ensure that the proportion of mutant mtDNA diagnosed in the biopsied cell gives an accurate indication of the mutant load in the remaining embryo. A heteroplasmic mouse model, carrying NZB and BALB mtDNA genotypes, was used to study the relative proportions of each mtDNA genotype in the ooplasm and first polar body of mature oocytes, and between blastomeres of early cleavage stage embryos. The levels of heteroplasmy varied widely in the gametes compared with the maternal genotype. However, the distribution of the two mtDNA genotypes was virtually identical between the ooplasm and polar body of a mature oocyte, and also between the blastomeres of each 2-, 4- and 6-8-cell embryo. Therefore, the level of heteroplasmy diagnosed from the polar body of an unfertilized oocyte or from a single blastomere of an embryo is representative of the level in the embryo as a whole. Reliable results were obtained from both polar bodies and blastomeres, but the efficiency of diagnosis was greater with blastomeres. We conclude that preimplantation genetic diagnosis is feasible for mtDNA diseases, although it should be approached with caution, as it is possible that transmission of some pathogenic mutations could behave in a different manner.     

多重置换扩增在植入前遗传学诊断中的应用及展望

多重置换扩增是一种新兴的全基因组扩增技术,能对单个细胞进行全基因扩增,产生大量的优质DNA,具有高扩增效率和高保真性等特点.多重置换扩增联合常规PCR已被成功用于植入前遗传学诊断,进一步扩展了后者的应用范围.     

Carrier-specific breakpoint-spanning DNA probes: an approach to preimplantation genetic diagnosis in interphase cells

Carriers of chromosomal inversions or other balanced rearrangements represent a significant fraction of patients in in-vitro fertilization (IVF) programmes due to recurrent reproductive problems. In most cases, chromosomal imbalance in fertilized oocytes is incompatible with embryo survival leading to increased rates of spontaneous abortions. Assuming that a fraction of the germ cells is karyotypically normal, these patients would greatly benefit from efficient procedures for generation and use of breakpoint-specific DNA hybridization probes in preconception and preimplantation genetic diagnosis (PGD). We describe the generation of such patient-specific probes to discriminate between normal and aberrant chromosomes in interphase cells. First, a large insert DNA library was screened for probes that bind adjacent to the chromosomal breakpoints or span them. Then, probe and hybridization parameters were optimized using white blood cells from the carrier to increase in hybridization signal intensity and contrast. Finally, the probes were tested on target cells (typically polar bodies or blastomeres) and a decision about the colour labelling scheme was made, before the probes can be used for preconception or preimplantation genetic analysis. Thus, it was demonstrated that cells with known structural abnormalities could be detected, based on hybridization of breakpoint spanning yeast artificial chromosome (YAC) DNA probes in interphase cells.      

Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis

Previously the diagnosis of sex and cystic fibrosis status hasbeen studied on single cells using the polymerase chain reaction(PCR). It has been suggested that allelic drop-out (PCR failureof one allele) and/or preferential amplification (hypo-amplificationof one allele) may contribute to poor reliability and misdiagnosis,although this remains controversial as some reports suggestthat allelic drop-out does not occur. We investigated an improvedmethod of diagnosing sex and cystic fibrosis in single cellsusing a new technology (fluorescent PCR) to determine the baselevel of PCR artefacts (allelic drop-out and preferential amplification)which, in combination with improved sensitivity, should improvePCR reliability and accuracy. Fluorescent PCR gives high reliability(97%) and accuracy rates (97%) in somatic cells for both sexand cystic fibrosis diagnosis and its lower detection thresholdallows allelic drop-out and preferential amplification to beeasily distinguished. We also achieved high reliability andaccuracy in diagnosing cystic fibrosis in human blastomeres.This study confirms earlier reports of both allelic drop-outand preferential amplification in single cell analysis. We demonstratethat both allelic drop-out and preferential amplification occurin somatic cells and suggest these are separate phenomena. Preferentialamplification appeared common in single cell PCR while allelicdropout apparently occurred at random in each allele. Preferentialamplification was mainly amplification of the larger allele.We suggest that some inaccuracy/misdiagnosis may be due to bothpreferential amplification as well as allelic drop-out. Otherfindings were variability in drop-out between PCR and that amplificationof signals from human blastomeres may be linked to embryo quality.We suggest that allelic drop-out is dependent on the numberof cells within the sample. allelic drop-out/cystic fibrosis/preferential amplification/preimplantation diagnosis/sexing     

A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells

A sensitive immunoassay is described for the detection of idiotype- and isotype-specific antibody-secreting cells (ASC), based upon the well established principles of ELISA. Single cell suspensions containing ASC are incubated on solid phase to which specific antigen has been chemically conjugated. Antibody attaches to the latter within the immediate microenvironment of the ASC, producing localized zones of bound antibody which are subsequently developed as visual 'spots' in the ELISA. This assay system has sensitivity and specificity at least equivalent to haemolytic plaque assays.     

Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease

Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.     

植入前遗传学诊断中卵裂球固定技术改进

目的改进植入前遗传学诊断卵裂球固定技术,减少卵裂球细胞丢失,获得更好的固定效率、更高的荧光原位杂交成功率,简化操作程序.方法体外授精治疗周期废弃胚胎的96个卵裂球细胞,分别用3种不同的固定技术进行固定,固定后使用X、Y着丝粒探针进行荧光原位杂交,比较其固定效率和杂交效率.结果改进方法固定率和杂交率均为100%.而两种传统技术的固定率分别为90%,83.3%;杂交率为80%,73.3%.结论这种新的固定卵裂球技术从根本上消除了卵裂球标本的丢失,使PGD结果更为可靠;同时简化了操作程序,值得推广.     

Single intragenic microsatellite preimplantation genetic diagnosis for cystic fibrosis provides positive allele identification of all CFTR genotypes for informative couples

This study is part of a strategy aimed at using fluorescent polymerase chain reaction (PCR) on informative genetic microsatellite markers as a diagnostic tool in preimplantation genetic diagnosis (PGD) of severe monogenic disease. Two couples, both of whom had previously had children who were compound heterozygote for severe cystic fibrosis mutations, were offered PGD using fluorescent PCR of the highly polymorphic cystic fibrosis transmembrane conductance regulator (CFTR) intragenic microsatellite marker IVS17bTA. Cleavage-stage embryo biopsy followed by PCR resulted in transfer of one unaffected carrier embryo for each couple. This approach eliminates the need for single cell multiplex PCR strategies to detect CF compound heterozygotes. It also provides a control of chromosome 7 ploidy in the blastomeres and a selection against allele dropout by positive detection of each CFTR copy of all genotypes in preimplantation embryos from genetically informative families.     

DNA amplification methods for diagnosis and epidemiological investigations of avian mycoplasmosis.

This review describes some applications of DNA amplification methods for diagnosis or epidemiological investigations of avian mycoplasmosis. Tests for direct detection of pathogenic mycoplasmas have been developed. Moreover, most avian mycoplasma species can be differentiated, according to their unique restriction fragment length polymorphism (RFLP) patterns generated after digestion of PCR products with different restriction enzymes. In order to characterize isolates below the species level, PCR-based subtyping methods have been introduced. One of them, arbitrarily primed-PCR, results in strain-specific arrays of DNA fragments that can distinguish even closely related strains of a given species. This method was successfully used to investigate the molecular epidemiology of vaccine strains and of Mycoplasma gallisepticum conjunctivitis in songbirds. Major issues in the development of DNA-amplification tests concern the selection of the appropriate target, specimen collection, DNA preparation and detection of amplification reaction inhibitors. Careful consideration to the design and work flow of the facility are necessary to avoid false-positive results.     

DNA amplification for the diagnosis of cat-scratch disease in small-quantity clinical specimens

Diagnosis of cat-scratch disease (CSD) by polymerase chain reaction (PCR) of lymph node fineneedle aspiration (FNA) and primary lesion specimens can be difficult owing to the minute amount of available material. A PCR assay specifically suited to test these specimens was developed. First, small-quantity (10 microL) samples were prepared from 17 CSD-positive and 16 CSD-negative specimens, and DNA extraction and amplification from these samples were compared using 3 methods. Sensitivity and specificity of PCR were 100% using material collected on glass microscope slides and by using Qiagen (Hilden, Germany) columns for DNA extraction. Then, this method was used to test 11 archival glass microscope slides of FNA (7 malignant neoplasms, 4 undiagnosed lymphadenitis) and 2 primary lesion specimens. Two of the 4 lymphadenitis samples and the 2 primary lesion specimens were PCR positive. The technique presented could facilitate CSD diagnosis from a wider range of clinical samples.     

HLA-DRB101 subtyping by allele-specific PCR amplification: A sensitive, specific and rapid technique

The two DR1-associated cellular specificities Dw1 and Dw20, as well as DR'Br' (Dw'BON'), cannot be unequivocally assigned by serological typing or restriction fragment length polymorphism (RFLP) analysis. We have developed and compared two polymerase chain reaction-based (PCR) typing methods for distinguishing these DRB1 alleles; allele-specific amplification of DRB1*01 alleles followed by an agarose gel electrophoresis detection step and group-specific DRB1*01 amplification followed by hybridization with sequence-specific oligonucleotide probes. The two typing strategies gave completely concordant results in the 33 DRB1*01-positive and the 46 DRB1*01-negative individuals and cell lines studied. No false-negative or false-positive typing results were obtained. All possible heterozygous combinations of the DRB1*0101-0103 alleles could be distinguished by both typing methods. DRB1*01 subtyping by allele-specific PCR amplification was performed in less than 3 hours, including PCR amplification, detection and interpretation steps. The technique will be a valuable complement to DR typing by serology and RFLP analysis. Allele-specific DRB1 amplifications or group-specific amplifications followed by directed allele-specific amplifications of DRB1 alleles, typing based on the absence or presence of amplified products, may well prove to be the technical innovation that will firmly establish PCR-based DR typing in routine clinical tissue typing.     

A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases

The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing.     

A rapid method for PCR amplification of DNA directly from cells fixed in Carnoy's fixative

We describe a method for rapid and efficient polymerase chain reaction (PCR) amplification of specific target DNA sequences directly from cells fixed in 3:1 methanol-acetic acid (Carnoy's fixative) for routine cytogenetic analysis. The fixed cells used had been stored at −20°C from a few weeks up to 6 years. Primer sets used correspond to loci on an autosome (retinoblastoma, RB1), as well as the X (Duchenne muscular dystrophy, DMD) and Y (sex-determining region of the Y, SRY) chromosomes. Sizes of amplified products were the expected 400, 251 and 609 bps, respectively. No differences in quality of amplification products were found between PCR templates obtained from fresh tissues or from cells fixed for varying lengths of time in Carnoy's fixative. This technique has the following advantages: (1) it allows retrospective studies of genetic disorders from archived specimens; (2) it requires only a limited number of cells; (3) it is rapid and simple; and (4) it avoids multistep procedures required in extraction of the DNA. © 1995 Wiley-Liss, Inc.     

A simple and rapid method for HLA-DRB and -DQB typing by digestion of PCR-amplified DNA with allele specific restriction endonucleases

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, which we previously reported as an efficient and convenient typing technique for accurate definition of the HLA-DQA1 and -DPB1 alleles, is now extended and applied to HLA-DRB and -DQB typing. The second exon of the HLA-DRB (B1 and B3 or B4) and DQB (B1 and B2) genes was selectively amplified from genomic DNAs of 70 HLA-homozygous B cell lines by PCR. Amplified DNAs were digested with the restriction endonucleases, which can recognize allelic variations specific for HLA-DR, -DQ, and -Dw allospecificities and then subjected to electrophoresis in polyacrylamide gel. Of DRB genes, FokI, HinfI, HhaI, HphI, KpnI and SacII were selected and the 20 different polymorphic patterns of the restriction fragments thus obtained were found to correlate with each HLA-DR and -Dw type defined by serological and cellular typing. Of the DQB genes, FokI, HaeIII, HhaI, RsaI and Sau3AI produced nine different polymorphic patterns of the restriction fragments, correlating with the HLA-DQ and -Dw types. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DR, -DQ and -Dw types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes.     

Rapid diagnosis of tuberculous lymphadenitis by amplification of mycobacterial DNA]

In patients with cervical lymph node swelling, prompt differential diagnosis of tuberculosis leads to an appropriate treatment. A method based on DNA amplification and hybridization for the rapid detection of Mycobacterium tuberculosis was used for demonstrating its specific DNA in cervical lymph nodes biopsied from seven patients. The DNA specific for Mycobacterium tuberculosis was detected in 5 patients. Four of them were histologically diagnosed as tuberculous lymphadenitis. In another case with necrotizing lesions but without granulomatous reactions, culture method subsequently revealed a positive result. In other 2 cases, the diagnosis of tuberculosis was promptly excluded by both this method and conventional methods (histologic diagnosis/direct microscopy). The rapid detection of Mycobacterium tuberculosis by amplification of mycobacterial DNA is helpful particularly in differential diagnosis of cervical lymphadenopathy.