Direct sequencing of tobacco chloroplast genome by the polymerase chain reaction

We have developed a polymerase chain reaction (PCR) method for sequencing of tobacco chloroplast genome. In a mixture containing chloroplast DNA, 5-end-labeled oligonucleotide primer, Taq DNA polymerase and reaction buffer, we were able to sequence a segment of chloroplast 16S rRNA gene. The results showed that the 750 bp of DNA sequenced were identical to the sequence reported, indicating that direct sequencing method that we have developed is useful for the sequencing of chloroplast genome. To analyze the chloroplast genome more rapidly in those in vitro grown plantlets, we also developed a simple method which is applicable for the amplifications and sequencing of chloroplast 16S rRNA fragment from either 0.15 g of tobacco leaf or stem tissue. The readable sequences obtained from the presented methods were consistent with the published sequence.     

Diagnosis of alpha 1-antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products.

We have compared sequencing of cloned "polymerase chain reaction" (PCR) products and the direct sequencing of PCR products in the examination of individuals from six families affected with alpha 1-antitrypsin (AAT) deficiency. In families where paternity was in question we confirmed consanguinity by DNA fingerprinting using a panel of locus-specific minisatellite probes. We demonstrate that direct sequencing of PCR amplification products is the method of choice for the absolutely specific diagnosis of AAT deficiency and can distinguish normals, heterozygotes and homozygotes in a single, rapid and facile assay. Furthermore, we demonstrate the reproducibility of the PCR and a rapid DNA isolation procedure. We have also shown that two loci can be simultaneously amplified and that the PCR product from each locus can be independently examined by direct DNA sequencing.     

Incorporation of 7-deaza dGTP during the amplification step in the polymerase chain reaction procedure improves subsequent DNA sequencing

An improved method for sequencing human genomic DNA amplified by the polymerase chain reaction (PCR) procedure is described. The portion of the genome investigated is the 383 nucleotide-long exon 2 of the human antithrombin III gene. Incorporation of the analogue of dGTP, 7-deaza-2'-deoxyguanosine-5'-triphosphate, during the amplification of exon 2 by PCR allowed for the elimination of recurrent artifacts obtained during sequencing of the amplified DNA by the dideoxyribonucleotide chain termination method.     

A simplified biochemistry for DNA sequencing

A simplified method of DNA sequencing by dideoxy chain termination is developed that approaches a single-step protocol. Utilizing the sequencing advantages contributed by a thermophilic polymerase and a guanine analog, stable sequencing reaction concentrates have been obtained that readily perform the entire sequencing reaction simply by adding prepared DNA to each of the four reaction concentrates required by this method. The mechanics and dynamics of these reactions have been investigated and the capacity of these reactions to withstand normal user variation is demonstrated. This study focuses on one form of this simplified method embodied in the FASTaq DNA sequencing kit.     

利用Taq DNA聚合酶对PCR产物直接进行顺序分析

Taq DNA聚合酶具有反应速度快、温度作用范围广及良好的续进性等特点,可视为一种理想的DNA顺序分析酶。本文首先对非对称性PCR扩增过程中单、双链DNA产物的积累情况进行了分析,然后采用标记延伸二步法,对Taq DNA聚合酶的性质及影响因素进行分析。为进一步改进Taq DNA聚合酶测序的方法,本反应建立了“Klenow-型”的直接掺入标记同位素测序法,即在反应液中加入与标记核苷酸相应的一定浓度的冷dNTP。此法不但解决了二步法中引物后部分DNA顺序无法读出的缺点,而且简化了反应步骤,亦能得到令人满意的顺序分析结果,每次可读出至少400碱基的序列。     

The "megaprimer" method of site-directed mutagenesis

We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.     

Direct sequencing of PCR products using the Maxam-Gilbert method

Direct sequencing of polymerase chain reaction (PCR) products by using the Maxam-Gilbert method is described. In this method, one of the primers is end labeled. Thus it is possible to sequence the reaction product directly following purification using this chemical method.     

Primer-independent RNA sequencing with bacteriophage phi6 RNA polymerase and chain terminators

Here we propose a new general method for directly determining RNA sequence based on the use of the RNA-dependent RNA polymerase from bacteriophage phi6 and the chain terminators (RdRP sequencing). The following properties of the polymerase render it appropriate for this application: (1) the phi6 polymerase can replicate a number of single-stranded RNA templates in vitro. (2) In contrast to the primer-dependent DNA polymerases utilized in the sequencing procedure by Sanger et al. (Proc Natl Acad Sci USA, 1977, 74:5463-5467), it initiates nascent strand synthesis without a primer, starting the polymerization on the very 3'-terminus of the template. (3) The polymerase can incorporate chain-terminating nucleotide analogs into the nascent RNA chain to produce a set of base-specific termination products. Consequently, 3' proximal or even complete sequence of many target RNA molecules can be rapidly deduced without prior sequence information. The new technique proved useful for sequencing several synthetic ssRNA templates. Furthermore, using genomic segments of the bluetongue virus we show that RdRP sequencing can also be applied to naturally occurring dsRNA templates. This suggests possible uses of the method in the RNA virus research and diagnostics.     

筛选转基因动物的新方法——PCR及其产物测序法

用特异性引物对肌球蛋白轻链2启动子(myosin light chain-2,MLC₂)-糜酶融合基因的转基因新生鼠鼠尾DNA进行PCR筛选, 低熔点琼脂糖凝胶电泳回收阳性样品PCR所扩出的DNA条带,纯化后用同一对引物中的一个进行单引物PCR测序,与所转外源基因序列比较,进一步确定整合有外源基因的阳性鼠.PCR及PCR 产物测序法检测转基因动物具有操作方便,灵敏度高及特异性强等优点.     

Affinity generation of single-stranded DNA for dideoxy sequencing following the polymerase chain reaction

A method to rapidly generate single stranded DNA for dideoxy sequencing following the polymerase chain reaction is described. By incorporating biotin in one of the amplification primers, we are able to physically separate the two DNA strands produced in the polymerase chain reaction. After amplification, the mixture is passed through a column containing streptavidin agarose. The strand produced by the biotinylated primer is bound in this matrix. The unbiotinylated strand is eluted with 0.2 N NaOH and sequenced by the dideoxy method. This method was utilized to sequence mitochondrial DNA from crude genomic DNA and to determine the sequences of four clones containing human mitochondrial DNA as a test of its accuracy. The use of biotin-facilitated separation permitted us to amplify and sequence DNA samples in a single day.     

The production of single-stranded DNA suitable for sequencing using the polymerase chain reaction

A simple and reliable procedure for the amplification of single-stranded DNA suitable for sequencing is described. This procedure employs the polymerase chain reaction and implements modifications pertaining to the purification of the double-stranded DNA product prior to single-stranded DNA amplification. The most consistent sequencing reactions are obtained when the double-stranded DNA product is purified by centrifugation with a microconcentrator prior to single-stranded DNA amplification and the overall amount of specific primers and number of cycles used, in both single-stranded and double-stranded DNA polymerase chain reactions, are reduced.     

快速检查寡聚DNA片段序列的方法

以质粒为模板,用待测寡聚DNA片段和通用测序引物进行PCR（聚合酶链式反应）,PCR片段经纯化后插入到pUC－18或pUC－19的多克隆位点中,然后用通用测序引物测定重组质粒上待测寡聚DNA片段,即可清晰、正确地知道它的序列．     

A New Solid-Phase Chemical DNA Sequencing Method Which Uses Streptavidin-Coated Magnetic Beads

To simplify the chemical DNA sequencing protocol, we developeda new solid-phase method which uses streptavidin-coated magneticbeads. This method is based on the finding that the biotinylatedDNA-streptavidin complex was stable under the conditions forsome chemical sequencing reactions. The 5'-biotinylated DNAgenerated by the polymerase chain reaction was first capturedby streptavidin-coated magnetic beads and then subjected toa set of simplified chemical sequencing reactions on the beadsat room temperature. Followed by the piperidine cleavage reaction,the products were resolved by gel electrophoresis, transferredonto a nylon membrane and visualized by chemiluminescent detection.As a consequence, highquality sequencing ladders were obtained,due to complete removal of contaminating chemicals, withoutthe time-consuming precipitation/centrifugation steps used inthe conventional chemical sequencing protocol     

Identification of enterohepatic Helicobacter species by restriction fragment-length polymorphism analysis of the 16S rRNA gene

Background. Restriction fragment-length polymorphism (RFLP) analysis of a 1,200-bp polymerase chain reaction–amplified DNA fragment of gene coding for 16S rRNA was used to generate restriction profiles of 11 enterohepatic Helicobacter spp. isolated from various animals and humans.
Methods. The amplicon from each Helicobacter sp. was digested with four restriction endonucleases: Alu I, Hinf I, Hha I, and Dde I. Alu I digestion produced five patterns that were useful for initial differentiation.
Results. Most Helicobacter spp. isolated from rodents had the same RFLP profiles by Alu I digestion (except H. rodentium and H. cholecystus ), but they had different RFLP profiles by Hha I digestion. Only H. bilis and " H. rappini" mouse isolates could not be readily distinguished by the polymerase chain reaction-RFLP method. However, these two species can be distinguished using H. bilis specific primers. Some of the Helicobacter spp. have an intervening sequence in their 16S rRNA gene, which changes the RFLP patterns; in these cases, sequencing is the preferred method to make an appropriate diagnosis.
Conclusions. The RFLP method used in this study was straightforward and rapid and should prove useful as an adjunct for identification and classification of multiple enterohepatic Helicobacter spp.     

Detection of the Cryptosporidium parvum "human" genotype in a dugong (Dugong dugon)

The Cryptosporidium "human" genotype was identified in a paraffin-embedded tissue section from a dugong (Dugong dugon) by 2 independent laboratories. DNA sequencing and polymerase chain reaction/restriction fragment length polymorphism analysis of the 18S ribosomal RNA gene and the acetyl CoA synthethase gene clearly identified the genotype as that of the Cryptosporidium variant that infects humans. This is the first report of the human Cryptosporidium genotype in a nonprimate host.     

α-1-Antichymotrypsin variant detected by PCR-single strand conformation polymorphism (PCR-SSCP) and direct sequencing

A new mutant -1-antichymotrypsin (variant ACT) was found by polymerase chain reaction single strand conformation polymorphism and direct sequencing. In this variant ACT, two bases (AA) were deleted from codon 391. This resulted in a different amino acid sequence downstream of the deletion point, elongating the peptide chain by 10 amino acids.     

Direct sequencing of long polymerase chain reaction fragments

Direct sequencing of polymerase chain reaction (PCR)-generated templates is a commonly used technique in molecular biology laboratories. We describe an improved method for direct sequencing of PCR fragments longer than 20 kb obtained with a commercial mixture ofTaq andPwo DNA polymerases. The sequencing protocol was optimized for an automated infrared DNA sequencer, consistently yielding long reads (500–600 bases).     

一种弗兰克氏菌分种新方法的探讨

弗兰克氏菌是非豆科植物共生固氮菌,目前已发现200多种。因其生长缓慢,许多传统的分类方法对它不合适,所以弗兰克氏菌的分类工作仍处于十分混乱的阶段。目前世界上公认弗兰克氏菌是放线菌的一个属,但没有确定的种名,寻找合适的分种方法十分迫切。我们选择16S-23S rRNA基因间隔区的序列作为划分弗兰克氏菌种的研究对象,现将结果简报如下。 1 材料和方法 1.1 菌种 101、114、8201,2129菌株分离自云南省西双版纳地区,101和114菌株是从木麻黄根瘤中分离到的,8201和2129菌株分别来自赤杨和杨梅根瘤。ArI_4和PtI_1菌株分别来自美国赤杨和潘尔稀根瘤。 1.2 DNA的提取     

Dideoxy sequencing of double-stranded DNA from poly(A)-extended 3'-ends

A simple method has been developed for sequencing double-stranded DNA by the chain termination method. The DNA to be sequenced is cut with a restriction enzyme that leaves a 3'-overhang which is extended by terminal deoxynucleotidyltransferase with limiting amounts of dATP. The sequencing reaction is then primed with an oligo(dT) primer which has a base pair "anchor" complementary to the overhang generated by the restriction enzyme. The method presented here eliminates the need for subcloning of the DNA or sequencing by chemical modification. Furthermore, sequences of more than 300 nucleotides are obtained from any 3'-overhanging restriction end.