DNA polymerase kappa microsatellite synthesis: Two distinct mechanisms of slippage‐mediated errors

Microsatellite tandem repeats are frequent sites of strand slippage mutagenesis in the human genome. Microsatellite mutations often occur as insertion/deletion of a repeat motif (unit‐based indels), and increase in frequency with increasing repeat length after a threshold is reached. We recently demonstrated that DNA polymerase κ (Pol κ) produces fewer unit‐based indel errors within dinucleotide microsatellites than does polymerase δ. Here, we examined human Pol κ's error profile within microsatellite alleles of varying sequence composition and length, using an in vitro HSV‐tk gap‐filling assay. We observed that Pol κ displays relatively accurate synthesis for unit‐based indels, using di‐ and tetranucleotide repeat templates longer than the threshold length. We observed an abrupt increase in the unit‐based indel frequency when the total microsatellite length exceeds 28 nucleotides, suggesting that extended Pol κ protein–DNA interactions enhance fidelity of the enzyme when synthesizing these microsatellite alleles. In contrast, Pol κ is error‐prone within the HSV‐tk coding sequence, producing frequent single‐base errors in a manner that is highly biased with regard to sequence context. Single‐nucleotide errors are also created by Pol κ within di‐ and tetranucleotide repeats, independently of the microsatellite allele length and at a frequency per nucleotide similar to the frequency of single base errors within the coding sequence. These single‐base errors represent the mutational signature of Pol κ, and we propose them a mechanism independent of homology‐stabilized slippage. Pol κ's dual fidelity nature provides a unique research tool to explore the distinct mechanisms of slippage‐mediated mutagenesis.Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

Identification of the parental origin of polysomy in two 49,XXXXY cases

The parental origin and mechanism of formation of polysomy X were studied in two polysomic cases, using four X-linked restriction fragment length polymorphisms, three (CA)_n dinucleotide repeat sequences and one variable number tandem repeat (VNTR) locus as genetic markers. A nonradioactive technique based on the hybridization of the polymerase chain reaction (PCR) product was developed for the analysis of dinucleotide repeats. Segregation analysis using different nonradioactive approaches based on the PCR, revealed that all four X chromosomes were of maternal origin. These data provide additional evidence of an identical mechanism of successive nondisjunctions in maternal meiosis I and II.     

Characterization of large CTG repeat expansions in myotonic dystrophy alleles using PCR

The CTG trinucleotide repeat expansions that are associated with myotonic dystrophy can be up to several thousand repeat units in length. We have developed a PCR protocol that has the potential to amplify mutant alleles with very large numbers of CTG repeats. The amplification uses the rTth DNA polymerase, XL system for long PCR targets together with primers which do not closely flank the repeat region and partial substitution of 7-deaza-dGTP for dGTP. Alleles containing up to approximately 800 CTG repeats were detected directly in agarose gels stained with ethidium bromide. Larger CTG repeat expansions required Southern blot transfer and detection with a repeat sequence probe; using this method, alleles containing up to approximately 2700 CTG repeats were detected. The PCR-based method described here was comparable to previous Southern blots of EcoRI-restriction digested genomic DNA in both the approximate size and heterogeneity of mutant alleles detected, but provided more precise sizes of the CTG repeat expansions than the restriction digest approach. This PCR protocol could potentially simplify current mutation detection protocols in the molecular diagnosis of myotonic dystrophy, and facilitate molecular studies of the disease. © 1996 Wiley-Liss, Inc.     

Regional reproducibility of microsatellite instability in sporadic colorectal cancer.

The reproducibility of microsatellite instability from different regions of the same sporadic colon cancer has not been addressed. We therefore microdissected and extracted DNA from three to nine separate regions of 13 highly unstable sporadic colon cancers. Each region was then evaluated by polymerase chain reaction amplification of 17 microsatellites: 10 tetranucleotide repeats, 2 noncoding mononucleotide repeats (BAT-26 and BAT-40), and 5 coding mononucleotide repeats (TGFBRII, BAX, MSH3, MSH6, IGFIIR). Microsatellite instability showed 100% regional reproducibility with respect to either the panel of 10 tetranucleotide repeats or BAT-26, and nearly 100% reproducibility with BAT-40, although regional variation in the percent instability and the size of unstable alleles was present. TGFBRII was more frequently mutated than any other coding mononucleotide repeat; frame shifts in this gene were identified in nearly every region of every tumor. Each of the five coding repeats showed regional variability in at least one tumor, and 10 of the 13 tumors showed variability with at least one coding repeat. This variability took the form of different mutant alleles (TGFBRII, BAX, MSH3) or mutations present in some but not all regions of a tumor (MSH6, IGFIIR, BAX, MSH3). We conclude that the regional reproducibility of generalized microsatellite instability as measured by noncoding repeats indicates that sampling is not a problem in these highly unstable tumors, and that the mismatch repair deficiency phenotype is acquired in the very late adenoma stage or early cancer stage of sporadic colonic tumorigenesis. The high frequency of TGFBRII mutations is consistent with acquisition of these mutations at a similar stage of tumorigenesis. The regional variability with respect to the presence or absence of a mutation in the other four coding mononucleotide repeats could lead to sampling error and is consistent with a somewhat later time of acquisition of these mutations. Genes Chromosomes Cancer 26:106-114, 1999.     

Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.     

Telomeric motifs are present in a highly repetitive element in the Plasmodium berghei genome

Using as probes the subfragments of the telomeric sequence previously cloned by us from Plasmodium berghei DNA, we identified and cloned a 2.3 kb repeat, largely overlapping the original telomeric insert. Restriction mapping indicated that cloned inserts (2.3 kb in length) represented circularly permutated versions of a rather well conserved repeated element, at least in part organized in tandem. The 2.3 kb repeat family with a copy number of about 300 occupies about 4% of the whole genome. The copies are unevenly distributed among the chromosome-sized molecules revealed by pulsed field gradient electrophoresis. Complete sequence determination of the 2.3 kb element revealed that telomere-related motifs are present with a characteristic pattern in a set of tandem repeats, 27 bp long. The perfect conservation of these motifs as well as the pattern of chromosomal distribution suggest that we are dealing with a specialised structure subject to selective mechanisms of amplification and maintenance.     

Minisatellite variant repeat (MVR) mapping: analysis of 'null' repeat units at D1S8.

Minisatellite variant repeat mapping by PCR (MVR-PCR) is a new approach to studying variation in human DNA which analyses interspersion patterns of variant repeats within minisatellite arrays. MVR-PCR has been applied to the hypervariable human minisatellite D1S8 which contains two major classes of variant 29bp repeat units designated a-type and t-type. The MVR-PCR assay uses a- or t-type specific primers, together with an amplimer at a fixed site in the DNA flanking the minisatellite, to reveal the interspersion patterns of variant repeats along an allele. Extreme levels of variation are seen both in the internal structures of individual alleles and in the digital code generated from the two superimposed alleles in total genomic DNA. However, occasional repeat units fail to amplify in MVR-PCR, signifying the existence of further repeat sequence variants termed 'null' or O-type repeats. Although not significant in individual identification, correct genotyping of null repeats is important when using MVR digital codes in parentage analysis. We have therefore characterised these null repeats and show that most null repeats share a common variant repeat sequence. We discuss the possible origins of null repeats and their application to paternity testing and the analysis of minisatellite evolution.     

Standardization of PCR amplification for fragile X trinucleotide repeat measurements

To provide the clinical diagnostics community with accurate protocols and measurements for the detection of genetic disorders, we have established a quantitative measurement program for trinucleotide repeats associated with human disease. In this study, we have focused on the triplet repeat associated with fragile X syndrome. Five cell lines obtained from the Coriell Cell Repository were analyzed after polymerase chain reaction (PCR) amplification and size separation. These cell lines were reported to contain CGG repeat elements (ranging from 29 to 110 repeats). Our initial measurements focused on measurement variability: (a) between slab-PAGE and capillary (CE) separation systems (b) interlane variability (slab-PAGE) (c) intergel variability, and (d) variability associated with amplification. Samples were run in triplicate for all measurements, and the analysis performed using Gene Scan analysis software. The repeat sizes were verified by DNA sequence analyzes. The standard deviations for interlane measurements in slab-gels ranged from 0.05 to 0.35. There was also little variation in size measurements performed on different gels and among PCR amplifications. The CGG repeat measurements performed by capillary electrophoresis were more precise, with standard deviations ranging from 0.02 to 0.29. The slab-PAGE and CE size measurements were in agreement except for the pre-mutation alleles, which yielded significantly smaller sizes by CE.     

Genetic variant characterization in intron 4 of the surfactant protein B gene

Genetic variants in intron 4 of the surfactant protein B gene SFTPB have been associated with pulmonary morbidity in newborn infants and adults. Genetic variant discovery in intron 4 requires high fidelity polymerase amplification due to a variable number of intermotif dinucleotide repeats and reliable characterization of alleles genetically distinct due to insertion, deletion, or both of 11 conserved sequence motifs. To optimize genetic variant discovery, we selected a high fidelity polymerase enzyme by replicate amplification and compared automated sequencing with agarose gel electrophoresis for all variant alleles (N=68) in a cohort of Missouri infants with (N=187) and without (N=53) respiratory distress. We identified and characterized six new alleles based on sequence motifs and two pairs of variant alleles with similar mobilities by agarose gel electrophoresis but distinct motif sequences. We confirmed uniformity of invariant alleles by sequencing (N=77). Logistic regression using race and intron 4 genotype for infants > or = 35 weeks gestation suggested that the invariant allele was independently associated with increased risk of respiratory distress (OR for the invariant allele 2.7, 95% CI 1.0-7.2). Reliable characterization of genetic variants in intron 4 requires both a high fidelity polymerase and sequencing of variant alleles.     

Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtiter wells.

We developed an improved microtiter-based assay for the detection of polymerase chain reaction (PCR)-amplified DNA sequences. The synthetic DNA sequences used to prime the PCR were labeled with biotin at their 5' ends so that the specific PCR product was labeled with biotin. Following amplification, an aliquot of the PCR product was denatured and hybridized to a capture DNA sequence immobilized in a microtiter well. The capture sequence was complementary to a portion of the sequence between the primers, so that only extended primers were captured. The captured PCR product was detected colorimetrically by using a streptavidin-peroxidase conjugate and tetramethylbenzidine substrate.     

Genome analysis of filamentous fungi: identification and characterization of an unusual GT-rich minisatellite in the ascomycete Podospora anserina

A genomic DNA fragment, PaGT7-5, of Podospora anserina was cloned that contains three different repetitive sequence motifs including a minisatellite with an unusual structure. This element, PaMin1, consists of ten copies of a 16 bp repeat unit with five GT dinucleotide repeats. Adjacent to PaMin1, a short poly(GT) stretch and four repeats of a 12 bp sequence were identified. Six alleles which differ in the number of the minisatellite unit were demonstrated in 18 different P. anserina strains. The flanking sequences of PaMin1 did not display any sequence alterations. A PCR analysis of total DNA from cultures of different age revealed no sequence changes, indicating that this repetitive DNA remains stable during aging. Southern-blot hybridization experiments of DNA from different strains detected only minor fragment length polymorphisms in the immediate vicinity of PaMin1. In contrast, major polymorphisms were observed at a greater distance from the locus indicating that this locus can be used as an informative probe to discriminate between different P. anserina strains. Received: 12 March / 24 May 1998     

Integrins and adhesion molecules: Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage-stage and blastocyst-stage human embryos

Human embryos have been biopsied at either the cleavage or theblastocyst stage of development. One to two blastomeres wereremoved from cleavage-stage embryos and 2–6 cells fromblastocysts. The biopsy specimens were subjected to gene amplificationby the polymerase chain reaction (PCR) and a comparison madeof amplification efficiencies of two unique target sequences,one located within the -globin gene and containing the sickle-celllocus and the other a polymorphic dinucleotide repeat. Whenthe cleavage-stage biopsy sample consisted of an intact blastomerewith a clearly discernible nucleus, an amplification efficiencyof 89% was achieved for each target locus. This was similarto that achieved with cleavage-stage biopsy samples consistingof two blastomeres or with blastocyst biopsy samples consistingof 2–3 trophectoderm cells. When biopsy samples consistedof four or more trophectoderm cells, both target loci were amplifiedin all samples tested. When the biopsy sample was heterozygousat the dinucleotide repeat locus and the biopsy consisted ofone or more intact cells with a clearly discernible nucleus,both alleles were amplified in >80% of biopsy samples. Whenfour or more trophectoderm cells were used for the PCR, bothalleles were amplified in all heterozygous samples. Target sequenceswere never amplified from biopsy samples which lysed prior totransfer into the reaction tube. Analysis of DNA fragments amplifiedfrom the dinucleotide repeat locus indicated that in most casesfaithful amplification of biopsy DNA template had taken place.However, in one case, fragments were identified which couldnot have resulted from the amplification of embryonic sequencesalone, indicating that contamination with extraneous DNA mayhave taken place. The significance of this finding for therapeuticpreimplantation genetic diagnosis is discussed.     

No evidence for BCL10 mutation in endometrial cancers with microsatellite instability

Previous reports have suggested that the mononucleotide repeats in BCL10 frequently are mutated in both hematologic malignancies and solid tumors. We set out to determine whether these repeats, like simple repeat sequences in other genes, are a target for mutation in endometrial cancers with defective DNA mismatch repair. Primary endometrial cancers (n = 42) and endometrial cancer cell lines (n=5) with microsatellite instability (MSI) were investigated. BCL10 exons 2 and 3 were amplified by PCR and evaluated for mutation using denaturing high-performance liquid chromatography (DHPLC) and single stand conformational variant (SSCV) analysis. Variants were directly sequenced. No BCL10 mutations were detected in exons 2 or 3 by DHPLC or SSCV. A polymorphism in exon 3 (638G-->A) was seen in 4/42 (9.5%) MSI-positive endometrial cancers and 0/5 MSI-positive endometrial cancer cell lines. Thus, mutation in the mononucleotide repeat tracts of BCL10 is not a feature of endometrial cancers with defective DNA mismatch repair.     

DNA template amplification genetic studies on archival human preimplantation biopsied microtubed cell samples

Biopsied cell samples can remain in storage following an initial, unequivocally successful preimplantation genetic diagnosis (PGD). The fidelity of the DNA template for polymerase chain reaction (PCR) amplification of microtubed human preimplantation embryonic cells stored at -70 degrees C for extended periods of time (6 months to 4 years) has been assessed. PCR protocols and specific nested primer sets for the beta-globin, ZP3 and CA repeat motif successfully used for previous human embryo PGD research were employed for these studies. The results show that the DNA template of microtubed biopsied blastomere and mural trophectoderm cell samples stored for periods of up to 4 years may be successfully amplified by PCR. Specific gene sequences were able to be analysed at the 1-2 or 3-5 biopsied cell level with a 71-100% success rate. Analysis of DNA fragments amplified from the CA dinucleotide repeat locus showed that in 8/9 samples both alleles were amplified at the cellular level. No DNA contamination was detected in the stored microtubed samples, or in the experimental controls.      

Quasimonomorphic mononucleotide repeats for high-level microsatellite instability analysis

Microsatellite instability (MSI) analysis is becoming more and more important to detect sporadic primary tumors of the MSI phenotype as well as in helping to determine Hereditary Non-Polyposis Colorectal Cancer (HNPCC) cases. After some years of conflicting data due to the absence of consensus markers for the MSI phenotype, a meeting held in Bethesda to clarify the situation proposed a set of 5 microsatellites (2 mononucleotide repeats and 3 dinucleotide repeats) to determine MSI tumors. A second Bethesda consensus meeting was held at the end of 2002. It was discussed here that the 1998 microsatellite panel could underestimate high-level MSI tumors and overestimate low-level MSI tumors. Amongst the suggested changes was the exclusive use of mononucleotide repeats in place of dinucleotide repeats. We have already proposed a pentaplex MSI screening test comprising 5 quasimonomorphic mononucleotide repeats. This article compares the advantages of mono or dinucleotide repeats in determining microsatellite instability.     

Routine clinical application of the FRAXA Pfu PCR assay: limits and utility

Fragile X genotype is characterized by the excessive amplification of an unstable region of DNA: a trinucleotide repeat CGG of variable copy number present in the FRAXA locus. Methods based on polymerase chain reaction (PCR) amplification of the CGG repeat region could facilitate the development of a rapid screening assay. Unfortunately, amplification across CGG repeats can be inefficient and unreliable due to their 100% G+C base composition. The utility of the exonuclease-deficient Pfu polymerase for amplification and detection of the CGG repeats at the FRAXA locus has been reported. In the present study we analysed the utility of a Pfu PCR assay as a rapid initial screening method to rule out a diagnosis of fragile X syndrome in males with mental retardation. Affected males did not show any amplification products or a smear of amplification products between 350 and 550 bp. Only 10% of affected male samples did not show any amplification products, while the vast majority showed the amplification smear. The amplification smears represent a serious drawback of the method, since they cannot be distinguished from the amplification products of normal samples after separation in 1 % agarose gel. Several modifications of the PCR conditions were attempted to eliminate this problem, but none was appropriate for clinical applications. However, the problem was easily solved by using a higher resolution electro-phoretic system that allows a clear distinction of normal bands from pathological smears. We tested the specificity of the Pfu PCR assay, followed by an improved MetaPhor gel electrophoretic separation of PCR products, on 50 samples from normal males and 24 samples from affected males. The results showed that this method is a rapid, sensitive and specific assay for the exclusion of fragile X syndrome diagnosis in mentally retarded males.     

A quantitative method to detect HBV cccDNA by chimeric primer and real-time polymerase chain reaction

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), is a continuous double chain ring, while other DNA forms contain a gap on each chain at the position of direct repeat 1 sequence (DR1) and direct repeat 2 sequence (DR2), respectively. At present it is still difficult to detect with high sensitivity and specificity and quantify the HBV cccDNA pool in the nucleus of the hepatocyte. A chimeric primer was designated in which segment A near 3' end is complementary to HBV plus strand just before the DR2 region gap, and segment B near 5' end is consensus to part of the human immunodefficient virus genomic sequence, without homogenetic relationship to HBV genome. Promoted by taq DNA polymerase, a single nucleotide strand is elongated from chimeric primer generated by HBV cccDNA. In contrast, other HBV DNA forms do not produce a single nucleotide strand due to the cessation of elongation reaction at the DR2 gap. The newly formed single nucleotide strand is subsequently amplified by a new polymerase chain reaction system (PCR), in which a primer, identical to chimeric primer segments B, is used to ensure specific amplification, avoiding other HBV DNA format inference. In addition, a taqman probe was used in the PCR system to report the detection signal, and for constructing a standard curve between cycle threshold (Ct) value and the template quantity. This technique proved to be effective for rapid and sensitive detection and quantitation of HBV cccDNA with high specificity and efficacy.     

Reliability and accuracy of polymerase chain reaction amplification of two unique target sequences from biopsies of cleavage-stage and blastocyst-stage human embryos

Human embryos have been biopsied at either the cleavage or theblastocyst stage of development. One to two blastomeres wereremoved from cleavage-stage embryos and 2–6 cells fromblastocysts. The biopsy specimens were subjected to gene amplificationby the polymerase chain reaction (PCR) and a comparison madeof amplification efficiencies of two unique target sequences,one located within the ß-globin gene and containingthe sickle-cell locus and the other a polymorphic dinucleotiderepeat. When the cleavage-stage biopsy sample consisted of anintact blastomere with a clearly discernible nucleus, an amplificationefficiency of 89% was achieved for each target locus. This wassimilar to that achieved with cleavage-stage biopsy samplesconsisting of two blastomeres or with blastocyst biopsy samplesconsisting of 2–3 trophectoderm cells. When biopsy samplesconsisted of four or more trophectoderm cells, both target lociwere amplified in all samples tested. When the biopsy samplewas heterozygous at the dinucleotide repeat locus and the biopsyconsisted of one or more intact cells with a clearly discerniblenucleus, both alleles were amplified in >80% of biopsy samples.When four or more trophectoderm cells were used for the PCR,both alleles were amplified in all heterozygous samples. Targetsequences were never amplified from biopsy samples which lysedprior to transfer into the reaction tube. Analysis of DNA fragmentsamplified from the dinucleotide repeat locus indicated thatin most cases faithful amplification of biopsy DNA templatehad taken place. However, in one case, fragments were identifiedwhich could not have resulted from the amplification of embryonicsequences alone, indicating that contamination with extraneousDNA may have taken place. The significance of this finding fortherapeutic preimplantation genetic diagnosis is discussed. ß-globin/CA repeat/embryo biopsy/polymerase chain reaction