一种快速、简便、高效cDNA合成及克隆策略

以鱼呼肠孤病毒双链ＲＮＡ为模板,建立一种快速、简便、高效的ｃＤＮＡ合成及克隆策略。在一定量模板条件下,采用随机六聚体为引物合成ｃＤＮＡ第一链。以退火方式形成双链ｃＤＮＡ,直接通过琼脂糖凝胶电泳检测其ｃＤＮＡ合成产量。双链ｃＤＮＡ经两端补齐后,平头连接于具有阳性选择标记的载体中,以高效电转化的方式进行快速克隆。     

Second-strand cDNA synthesis with E. coli DNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect of E. coli DNA ligase

A simple method for generating cDNA libraries has been described (1) in which RNase H-DNA polymerase I-mediated second-strand cDNA synthesis primes from an RNA oligonucleotide derived from the 5' (capped) end of mRNA. The size of this oligonucleotide and the fate of the information corresponding to the RNA during subsequent cloning have not been established. We show here that the 5'-most RNA primer varies in length from 8 to 21 nucleotides, and that information corresponding to the length of the RNA primer is normally lost during cloning. A modification of the second-strand cDNA synthesis procedure is described which allows cloning of all, or almost all, of the primer sequence information. In addition, we show that the presence of E. coli DNA ligase during second-strand cDNA synthesis can increase the length of the cDNA clones obtained from long RNAs. Cloning by addition of linkers provides the greatest chance of obtaining near full-length cDNA clones from long mRNAs.     

Use of synthetic DNA in expression of foreign proteins

Synthetic DNAs and oligonucleotides, which can be prepared conveniently by combining chemical synthesis and enzymatic methods, have been used extensively in recombinant DNA research. Examples include total gene synthesis, probes for the isolation of specific genes from cDNA or genomic libraries, linkers containing specific restriction sites for cloning, primers for DNA and RNA sequencing, and primers for the construction of specific mutations (either deletion, insertion or point mutations) by oligonucleotide-directed site-specific mutagenesis.This article reviews recent advances in the chemical and enzymatic synthesis of oligo- and polynucleotides and the application of synthetic DNA to the expression of foreign proteins. The synthesis of genes, including structural genes and regulatory genes are reviewed. Oligonucleotide-directed site-specific mutagenesis and use of synthetic DNA to optimize foreign protein expression are also discussed.     

鲤鱼垂体mRNA反转录模板活性的研究

 本实验用不同方法研究鲤鱼垂体mRNA反转录为互补DNA的活性。用硫氰酸胍法,从垂体中提取总RNA,经过Oligo(dT)-纤维素柱层析,得到poly(A)~+RNA。 以mRNA为模板,30％反转录成单链cDNA。利用RNaseH-DNA聚合酶Ⅰ-E.coli DNA连接酶合成双链cDNA。单链cDNA拷贝成双链cDNA。经放射自显影分析,证明合成了全长cDNA。用水解法合成双链cDNA,大多数为不完整的双链cDNA。平末端的双链cDNA连接上Eco RI-linker经Sepharose-4B分离,收集大片段cDNA,与pUC 19载体相连接,经转化构建成10~5克隆/μg mRNA的cDNA文库。     

Directional random oligonucleotide primed (DROP) global amplification of cDNA: its application to subtractive cDNA cloning.

We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and the use of a biotinylated primer permits the separation of single-stranded cDNA. Isolation of these products from different cell types provides a renewable source of target single-stranded cDNA and driver RNA from limited cell numbers and we demonstrate their use for subtractive hybridisation cloning of differentially expressed cDNAs.     

Amine-modified random primers to label probes for DNA microarrays

DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 microg of total RNA or 2 microg of poly(A) RNA. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 microg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5' ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simple to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers.     

PCR fragmentation of DNA

A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.     

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.     

A modified procedure for PCR-based differential display and demonstration of use in plants for isolation of genes related to fruit ripening

We have modified and optimized PCR-based differential display for efficient identification and isolation of genes whose expression patterns are correlated with changes in growth, development, physiology, and/or environmental response. This protocol is general in nature and can be applied for analysis of virtually any plant tissues from which several μg of total RNA can be extracted. We report here the use of tomato fruit ripening as a model system in which to test and optimize differential display in plants. Specifically, mRNA from ripe, early breaker, mature green, and ethylene-treated mature green tomato fruit were examined to identify and distinguish non-ethylene-inducible from ethylene-inducible genes related to ripening. DNA-free total RNA was utilized as template for synthesis of first-strand complementary DNA using each of 12 possible 5′-T₁₁ XY-3′ anchor primers (X=A, C, or G; Y=A, C, G, or T). PCR amplification products of the resulting cDNA populations were generated via use of random primers in combination with the corresponding anchor primer employed for cDNA synthesis. We demonstrate that degenerative anchor primers are useful for making representative cDNA populations, but are not effective for representative display-PCR. cDNA, resulting from degenerative anchor primer synthesis, yielded substantially fewer ripening-related display-PCR products when amplified with the same degenerative anchor primer employed in cDNA synthesis, versus the corresponding set of specific anchor primers. Amplification products specific to ripe fruit cDNA were isolated directly from display gels, reamplified, cloned, and confirmed for ripening-related gene expression via RNA gel-blot analysis.     

Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioate-modified primers

Directional cloning of complementary DNA (cDNA) primed by oligo(dT) is commonly achieved by appending a restriction site to the primer, whereas the second strand is synthesized through the combined action of RNase H and Escherichia coli DNA polymerase I (PolI). Although random primers provide more uniform and complete coverage, directional cloning with the same strategy is highly inefficient. We report that phosphorothioate linkages protect the tail sequence appended to random primers from the 5′ → 3′ exonuclease activity of PolI. We present a simple strategy for constructing a random-primed cDNA library using the efficient, size-independent, and seamless In-Fusion cloning method instead of restriction enzymes.