Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA

A solid-phase approach is described for manual and automated sequencing of plasmid DNA obtained directly from bacterial colonies through the polymerase chain reaction. The DNA fragment is selectively immobilized to magnetic beads and after strand-specific elution, the eluted strand, as well as the remaining immobilized strand, is used for bidirectional dideoxy sequencing. The solid-phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The approach is exemplified by fluorescent sequencing of a cloned Streptomyces curacoi gene having a G + C content of more than 70%.     

The ligation amplification reaction (LAR)--amplification of specific DNA sequences using sequential rounds of template-dependent ligation

A novel DNA sequence detection method that utilizes the ligation of oligonucleotide pairs that are complementary to adjacent sites on appropriate DNA templates is described. The product is increased by either linear or exponential amplification using sequential rounds of template-dependent ligation. In the case of linear amplification, a single pair of oligonucleotides is ligated, the reaction is heated to dissociate the ligation product, and an additional round of ligation is performed. After n rounds there is a (1 + x) X n-fold amplification of product, where x is the efficiency of the ligation reaction. Exponential amplification utilizes two pairs of oligonucleotides, one complementary to the upper strand and one to the lower strand of a target sequence. The products of the ligation reaction serve as templates for subsequent rounds of ligation. In this case there is (1 + x)(n-1)-fold amplification of product after n rounds. A single base-pair mismatch between the annealed oligonucleotides and the template prevents ligation, thus allowing the distinction of single base-pair differences between DNA templates. At high template concentrations, the ligation reaction has an efficiency approaching 100%. In this report, we demonstrate the use of the ligation amplification reaction (LAR) to distinguish the normal from the sickle cell allele of the human beta-globin gene. We also report the use of LAR as a detection system for polymerase chain reaction-enriched DNA sequences.     

A strategy for producing single-stranded DNA in the polymerase chain reaction: A direct method for genomic sequecing

A strategy for synthesizing single-stranded DNA by the polymerase chain reaction method is explored and two protocols are developed. DNA produced with these methods can be sequenced using standard ³⁵S or ³²P dideoxy sequencing protocols. The methods allow rapid genetic screening and population genetic analysis of sequence variation.     

Whole Genome Amplification for Sequencing and Applications in Conservation Genetics

Abstract: We describe a method for rapidly amplifying whole genomes via a Phi29 DNA polymerase-mediated strand displacement reaction (SDR). Genomic amplification products derived from the SDR reaction resulted in high quantities of DNA suitable for polymerase chain reaction (PCR) amplification and sequencing of mitochondrial genomes. Control region sequences of DNA derived directly from PCR amplicons of extracted DNA were identical to those derived from PCR amplification of SDR genomic DNA. Effective SDR amplification and subsequent sequencing was successful across tissues sources ranging in age from 1 year to 19 years. Strand replacement reaction genomic amplification offers a means of obtaining large quantities of DNA from small amounts of tissue.     

A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing

A set of plasmid vectors was developed which allows fast sequencing by the chemical degradation method. These high-copy-number vectors are derivatives of the plasmid pUC8 containing different multiple-purpose cloning sites flanked by unique recognition sequences for the restriction enzymes BstEII, Tth111I and Eco81I as sites for end-labelling DNA. Due to their partially asymmetric recognition sequences, each of these three restriction sites can be singly end-labelled by a filling-in reaction with selected nucleotides. This allows easy single end-labelling of any cloned DNA fragment for sequencing by the chemical degradation method without any isolation and purification step after the labelling reaction. In addition, the nucleotide sequence of the complementary strand from the same end can be determined by the dideoxy chain termination procedure using the universal M13 primers. In most of the new vectors, the reading frame of the lacZ' gene is retained, allowing identification of cloned fragments.     

Polymerase chain reaction amplification of single-stranded DNA containing a base analog, 2-chloradenine.

By utilization of polymerase chain reaction techniques, single-stranded DNA of defined length and sequence containing a purine analog, 2-chloroadenine, in place of adenine was synthesized. This was accomplished by a combination of standard polymerase chain amplification reactions with Thermus aquaticus DNA polymerase in the presence of four normal deoxynucleoside triphosphates, M13 duplex DNA as template, and two primers to generate double-stranded DNA 118 bases in length. An asymmetric polymerase chain reaction, which produced an excess of single-stranded 98-base DNA, was then conducted with 2-chloro-2'-deoxy-adenosine 5'-triphosphate in place of dATP and with only one primer that annealed internal to the original two primers. Standard polymerase chain reaction techniques alone conducted in the presence of the analog as the fourth nucleotide did not produce duplex DNA that was modified within both strands. This asymmetric technique allows the incorporation of an altered nucleotide at specific sites into large quantities of single-stranded DNA without using chemical phosphoramidite synthesis procedures and circumvents the apparent inability of DNA polymerase to synthesize fully substituted double-stranded DNA during standard amplification reactions. The described method will permit the study of the effects of modified bases in template DNA on a variety of protein-DNA interactions and enzymes.     

Promoter-detection vectors for Escherichia coli with multiple useful features

Two promoter-detection vectors have been constructed which enable the cloning and characterization of promoters recognized by the RNA polymerase of Escherichia coli K-12. The intergenic region of phage M13 DNA, present in opposite orientations in the two vectors, permits the preparation of single-stranded DNA of either strand of the insert thus facilitating oligodeoxyribonucleotide heteroduplex mutagenesis and sequencing of both strands by the dideoxy method of chain termination. After mutagenesis, isolates can be screened for changed function by replica-plating colonies to plates containing XGal. Selected isolates can be characterized by nucleotide sequence analysis, to determine the change in structure, and by beta-galactosidase assays, thus measuring the effect of mutagenesis on promoter function. The vectors could also be used like other protein-fusion vectors.     

Enzymatic syntheses of DNA-silicas using DNA polymerase.

Oligothymidylic acids couple to an activated ester silica (N-hydroxysuccinimidyl-silica) only when they contain an added aminoalkyl group. Heteropolymeric oligomers containing other nucleotide bases were shown to also couple by way of the nucleotide base (adenine, cytosine, or guanine); however, when a heteropolymeric oligonucleotide also contains a 5'-aminoalkyl moiety, coupling by way of the latter is the favored reaction. When duplex hybrids of oligonucleotides are formed, the nucleotide bases are protected from chemical coupling. Coupling by way of nucleotide bases would be detrimental to some chromatography experiments. On the basis of these observations, two different procedures were developed to produce DNA-silicas in which a single strand of the DNA is coupled by only its 5'-terminus. In the first of these, the polymerase chain reaction was used with a 5'-aminoalkyl primer to make a duplex DNA with one strand containing the 5'-aminoalkyl group and the duplex DNA is then coupled to the activated ester silica. This yielded a silica containing about 0.17 nmol of a 242-mer per gram silica which bound only probes specific for the coupled strand. In the other procedure, a template DNA strand was poly(A) tailed and hybridized to (dT)18-silica. DNA polymerase I (Klenow large fragments) was then used to copy the template-specified sequence directly onto the 3'-terminus of the (dT)18. This procedure yielded about 1.2 to 2.7 nmol DNA copied/g of silica of a specific 21-mer sequence. The DNA-silica produced selectively hybridized only with complementary sequences and not with DNA lacking that sequence. Either of these procedures thus produces DNA-silicas from heteropolymeric DNA sequences with a predetermined, specific 5'-terminal site of attachment.     

Combination of amplification and post-amplification strategies to improve optical DNA sensing

The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.     

Strategies for automated sequencing of human mitochondrial DNA directly from PCR products.

A rapid, robust and sensitive method has been developed for the amplification and direct sequencing of human mitochondrial DNA. A 403-bp hypervariable segment was amplified by two successive rounds of nested PCR. This was then sequenced by the dideoxy chain termination method using dye-labeled universal sequencing primers in conjunction with an automated DNA sequencer. This paper describes the assessment of four different strategies for this amplification and sequencing process. Optimal results were obtained by immobilizing the biotinylated PCR product on Dynabeads followed by solid-phase sequencing with Sequenase. Degraded samples and those containing trace amounts of DNA such as extracts from hair shafts can be analyzed by this method.     

Application of nucleic acid amplification in clinical microbiology

The use of nucleic acid amplification methods in routine clinical microbiology laboratories is becoming increasingly widespread. The theory of polymerase chain reaction is described, including discussion of suitable microbal targets, extraction of nucleic acid from clinical samples, choice of primers, optimization of the process, laboratory design, contamination, and other problems as well as quality control. Other nucleic acid amplification methods such as ligase chain reaction, self-sustained sequence replication, strand displacement amplification, and branched DNA signal amplification are described and the choice of technology is discussed.     

Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies.

The whole genome amplification (WGA) protocol evaluated during this study, GenomiPhi DNA amplification kit, is a novel method that is not based on polymerase chain reaction but rather relies on the highly processive and high fidelity Phi29 DNA polymerase to replicate linear genomic DNA by multiple strand displacement amplification. As little as 1 ng of genomic DNA template is sufficient to produce microgram quantities of high molecular weight DNA. The question explored during this study is whether such a WGA method is appropriate to reliably replenish and even recover depleted DNA samples that can be used for downstream genetic analysis. A series of human DNA samples was tested in our laboratory and validated using such analytical methods as gene-specific polymerase chain reaction, direct sequencing, microsatellite marker analysis, and single nucleotide polymorphism allelic discrimination using TaqMan and Pyrosequencing chemistries. Although degraded genomic DNA is not a good template for Phi29 WGA, this method is a powerful tool to replenish depleted DNA stocks and to increase the amount of sample for which biological tissue availability is scarce. The testing performed during the validation phase of the study indicates no discernable difference between WGA samples and the original DNA templates. Thus, GenomiPhi WGA can be used to increase precious or depleted DNA stocks, thereby extending the life of a family-based linkage analysis project and increasing statistical power.     

Influence of neighboring base sequence on mutagenesis induced by in vitro misincorporation in the lacI gene of Escherichia coli.

Genetic and electrophoretic assays of misincorporation were used to assess the effect of DNA sequence on mutagenesis arising from in vitro DNA synthesis within the lacI gene of Escherichia coli. The viral strand of a derivative of phage M13 containing the entire lacI gene was annealed with a series of synthetic oligonucleotides complementary to the N-terminal region of the lacI gene. Each primer-template was incubated with E. coli DNA polymerase I (Klenow fragment) under conditions favoring misincorporation, wherein one of the 4 dNTPs was lacking ('minus' reaction) or present at very low concentration ('micro' reaction). The extent of elongation of each primer was assessed by gel electrophoresis, and lacI mutants arising during the misincorporation reactions were detected by a transfection assay in which i- base substitutions within the in vitro synthesized strand were selectively recovered by the use of uracil-containing templates. Direct dideoxy sequencing of the '-A' reaction products and sequence analysis of i- mutant progeny revealed a vast predominance of single and non-tandem multiple base transitions. The addition of small quantities of dATP to a '-A' reaction increased the mutation yield and broadened the distribution of base substitutions along the template. We detected a general bias towards increased base substitution at template positions flanked by G.C base pairs or 5'-pyrimidine, 3'-purine nearest neighbors, although considerable site-to-site variation in the occurrence of base substitutions was seen, even within identical nearest neighbor contexts.     

A novel primer extension method to detect the number of CAG repeats in the androgen receptor gene in families with X-linked spinal and bulbar muscular atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA), an adult-onset form of motor neuron disease, was recently reported to be caused by amplification of the CAG repeats in the androgen receptor gene. We report here a simple and rapid strategy to detect the precise number of the CAGs. After the DNA fragment containing the CAG repeats is amplified by the polymerase chain reaction, a primer extension is carried out; the extension of the end-labelled reverse primer adjacent to 3' end of CAG repeats stops at the first T after CAG repeats with the incorporation of dideoxy ATP in the reaction mixture. The resultant primer products are analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. This method could be quite useful to detect not only CAG repeats in SBMA but also other polymorphic dinucleotide and trinucleotide repeats.     

Single-molecule dilution and multiple displacement amplification for molecular haplotyping

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.     

PCR PRIMERS FOR THE AMPLIFICATION OF MITOCHONDRIAL SMALL SUBUNIT RIBOSOMAL DNA OF LICHEN-FORMING ASCOMYCETES

Abstract: Four primers for the amplification of mitochondrial DNA of lichen-forming ascomycetes are presented. The primers match the conserved regions U2, U4, and U6, respectively, of mitochondrial small subunit (SSU) ribosomal DNA (rDNA). Polymerase chain reaction using different combinations of the primers produced single amplification products from DNA of eight lichen-forming fungal species but did not amplify DNA of two axenic cultured algal species. The amplification product obtained from Lobaria pulmonaria was sequenced and the 894-bp sequence was compared with the mitochondrial SSU rDNA sequence of Podospora anserina. The two sequences revealed more than 76% identity in the conserved regions U3 to U5 demonstrating that we amplified mitochondrial DNA. The primers matching U2 and U6 yielded amplification products of 800–1000 bp depending on the species examined. The variation observed suggests that mitochondrial SSU rDNA may be useful for phylogenetic analyses of lichen-forming ascomycetes.     

A novel blocker-PCR method for detection of rare mutant alleles in the presence of an excess amount of normal DNA.

A novel polymerase chain reaction method was developed to preferentially amplify a segment of DNA containing a base substitution mutation. This technique uses a pair of dideoxynucleotide-labeled oligonucleotides (18 mers) of normal sequences as blockers located between the two primers. By virtue of a subtle difference in the melting temperature between the blocker-normal DNA and blocker-mutant DNA hybrids, the method allows preferential amplification of the mutant DNA. We used the human N-ras gene as a model. Two different types of N-ras mutations could be effectively amplified when they were present with an excess amount of normal DNA at a ratio of 1:10(3). Furthermore, the sensitivity was increased 10-fold by using single strand conformation polymorphism analysis for the amplified products, and mutant DNA was detected in the presence of a 10(4) times excess amount of normal DNA.     

Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA.

A specific segment of mitochondrial DNA from 18 people was examined by two methods of direct DNA sequencing. This segment includes a small noncoding region (V) shown before by restriction analysis to exhibit length polymorphism. All 11 of the human mtDNAs previously reported to have a deletion in this region proved to lack one of the two adjacent copies of a 9-base-pair sequence normally present in human mtDNAs. Phylogenetic analysis suggests that this deletion occurred only once during the evolution of modern types of human mtDNA and that it will be a valuable anthropological marker for peoples of East Asian origin. The one human mtDNA reported to have an addition in region V differs from the wild type by two mutations in the first copy of the 9-base-pair sequence: one transition and an addition of four cytosines, thereby producing a run of 11 cytosines. One of the direct DNA sequencing methods uses a single oligonucleotide primer to facilitate dideoxy sequencing from purified mtDNA templates. The second, more successful, method first amplifies this mtDNA segment enzymatically with two flanking primers (the "polymerase chain reaction") and then uses a third primer for DNA sequencing. This latter method, which works on the DNA extracted from small amounts of blood as well as on purified mtDNA, is shown to be a rapid means of defining sequence variants without purifying and cloning the same DNA segment from many individuals.