甲肝病毒减毒株(H2)RNA的全长RT-PCR扩增

通过RT-PCR技术扩增了甲肝病毒减毒株(H₂)全长RNA,并对长片段RT-PCR扩增进行了方法学上的探讨.采用抗血清特异沉淀病毒;盐酸胍-酸性酚、氯仿一步法分离纯化病毒RNA,可得到高质量的RNA样品;以此RNA为模板,在无RNA酶的逆转录酶作用下,合成单链cDNA;继续以此cDNA为模板,利用32 mer寡核苷酸引物, 在Taq和Deep Vent DNA多聚酶的作用下进行PCR扩增,得到7.4 kb的扩增产物.     

High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus.

A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.     

A strategy to study gene polymorphism by direct sequence analysis of cosmid clones and amplified genomic DNA

A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.     

The implications of using mutagenic primers in combination with Taq polymerase having proofreading activity.

Polymerases with proofreading activity provide high fidelity PCR amplifications. In this study we examined the consequences of using a Taq polymerase with proofreading activity, such as Optimase Taq polymerase, in combination with 4 different mutagenic reverse primers for the amplification of a 345-bp FII PCR product. The amplifications were performed with Optimase Taq polymerase (Transgenomic), and Taq DNA polymerase-recombinant (Invitrogen), without proofreading activity. Mutation screening was carried out by DHPLC and restriction fragment analysis. The usage of Optimase Taq polymerase results in complete reversion of the first and second mutated nucleotide introduced at the 3' end of the mutagenic reverse primer. It also partially reverses the missense nucleotide introduced in the third position of the mutagenic primer and leads to misleading DHPLC and restriction fragment analysis patterns. Nevertheless it cannot perform such an activity when an abnormal nucleotide is introduced in the fourth position.     

Study of transcription of the human cytochrome P450IID6 gene by polymerase chain reaction]

Polymerase chain reaction was used to study the expression of the drug metabolism gene. Primers complementary to the 2070-2090 and 2912-2930 sites within exons 4 and 6 of the gene CYP2D6 were synthesized. The amplification of the cDNA from total human liver mRNA was achieved. The length of the fragment obtained (238 bp) was in accordance with the distance between the primers binding sites in cDNA. The amplification of the DNA from the same source led to the longer fragment due to the presence of introns. The total RNA from the blood cells of the extensive metabolizers was shown to contain the mRNA transcribed from the CYP2D6 gene. The Taq polymerase reaction in the presence of cDNA derived from a poor metabolizer did not lead to the synthesis of the 238 bp fragment.     

Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus

The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction method for amplifying DNA. We report the cloning and expression of Taq DNA polymerase in Escherichia coli. From a lambda gt11:Taq library we identified a Taq DNA fragment encoding an epitope of Taq DNA polymerase via antibody probing. The fusion protein from the lambda gt11:Taq candidate selected an antibody from an anti-Taq polymerase polyclonal antiserum which reacted with Taq polymerase on Western blots. We used the lambda gt11 clone to identify Taq polymerase clones from a lambda Ch35:Taq library. The complete Taq DNA polymerase gene has 2499 base pairs. From the predicted 832-amino acid sequence of the Taq DNA polymerase gene, Taq DNA polymerase has significant similarity to E. coli DNA polymerase I. We subcloned and expressed appropriate portions of the insert from a lambda Ch35 library candidate to yield thermostable, active, truncated, or full-length forms of the protein in E. coli under control of the lac promoter.     

cDNA library construction from small amounts of unfractionated RNA: association of cDNA synthesis with polymerase chain reaction amplification.

We describe here a general and simple procedure for cDNA library construction making use of in vitro amplification of cDNA by polymerase chain reaction (PCR). The first-strand cDNA is synthesized from total RNA with a primer EcoRI-(dT)17 and oligo(dG) tailed. An oligonucleotide, EcoRI-BamHI-(dC)13, is used to prime the second-strand synthesis by the thermostable DNA polymerase of Thermus aquaticus. The double-stranded cDNA is then amplified directly by PCR. A study of the effect of the elongation time on the PCR products showed that a long extension time is necessary to overcome the size heterogeneity of the cDNA population. Starting from 1 microgram of total brain RNA, the products obtained ranged from 200 to more than 2000 bp. The presence of the myelin basic protein cDNA sequence was determined. A lambda gt10 library containing 2 x 10(6) clones was established with the amplified cDNA. No sequences originating from rRNA were detected by Southern blot analysis. The ability to produce representative cDNA libraries from minute amounts of total RNA by this protocol should have many applications to studies of gene expression in small amounts of tissues or cells.