cDNA末端快速扩增试剂盒研发进展

DNA扩增的方法有许多种,其中cDNA末端快速扩增（rapid amplification of cDNA ends,RACE）因其操作简单、成功率相对较高、重复性好,被广泛应用于真核生物基因全长的克隆与分析。本文比较了市售的RACE试剂盒所采用的模板制备策略及改进的扩增方法。     

用RACE技术快速扩增五指山猪来源的猪内源性反转录病毒3＇LTR

测定我国小型猪来源的猪内源性反转录病毒(PERV)3'LTR,以便于PERV全基因的克隆和分析。用cDNA末端快速扩增(RACE)技术,从五指山猪外周血淋巴细胞mRNA中扩增到PERV-3'LTR,并克隆入pGEM-Teasy载体,将阳性克隆进行序列测定和同源性分析。测序结果显示该克隆3'端的尾部有一个由12个A组成的poly(A)信号;其R区与PERV-MSL的R区(约64bp)基本一致,同源性分析表明其与PERV-MSL的3'LTR具有81%的序列同源性。说明成功扩增了我国五指山猪来源的PERV-3'LTR,将有利于PERV全基因的克隆。     

用RACE技术快速扩增五指山猪来源的猪内源性反转录病毒3'LTR

测定我国小型猪来源的猪内源性反转录病毒(PERV)3'LTR,以便于PERV全基因的克隆和分析.用cDNA末端快速扩增(RACE)技术,从五指山猪外周血淋巴细胞mRNA中扩增到PERV-3'LTR,并克隆入pGEM-T easy载体,将阳性克隆进行序列测定和同源性分析.测序结果显示该克隆3'端的尾部有一个由12个A组成的poly(A)信号;其R区与PERV-MSL的R区(约64 bp)基本一致,同源性分析表明其与PERV-MSL的3'LTR具有81%的序列同源性.说明成功扩增了我国五指山猪来源的PERV-3'LTR,将有利于PERV全基因的克隆.     

Isolation of cDNA clones for the human transferrin receptor.

A cDNA clone bank containing 30 000 clones was constructed from sucrose gradient-fractionated mRNA from human placenta. mRNA coding for transferrin receptor (TR) was enriched by polysome immuno-adsorbed chromatography with monospecific rabbit IgG and protein-A Sepharose. The library was screened for hybridisation to 32P-labelled cDNA synthesised from immunoselected TR mRNA and from poly(A)+ RNA of the polysome fraction that failed to bind to protein-A Sepharose. Plasmids isolated from colonies showing hybridisation only to the probe made from immunoselected mRNA were then subjected to hybrid selection. Two clones, pTR-48 and pTR-67, were able to hybridise the mRNA coding for the TR.     

Isolation and characterisation of a human calcitonin-gene-related-peptide receptor

We describe the identification and purification of a receptor for calcitonin-gene-related peptide (CGRP) from human term placenta, using lectin and beta-CGRP-Affigel affinity chromatography. The membrane-bound receptor has an estimated Mr of 240,000, as determined by cross-linking 125I-labelled alpha-CGRP (125I-alpha-CGRP) using discuccinimidyl suberate and SDS/polyacrylamide gel electrophoresis, or of 263,000, as judged by sucrose gradient centrifugation of the soluble partially purified native receptor preparation. Cross-linking studies with disuccinimidyl suberate and N-hydroxysuccinimidyl-4-azidobenzoate using membrane-solubilised, partially purified and CGRP-affinity-purified preparations, show a number of 125I-alpha-CGRP binding subunit(s) of Mr 62,000-68,000. Silver staining of the purified CGRP receptor preparation showed two distinct doublets in this plus a number of minor doublets of lower Mr. The receptor binds human beta-CGRP with greater affinity than alpha-CGRP, and showed little affinity for human calcitonin. Adsorption isotherms and Scatchard analysis of 125I-alpha-CGRP binding to the membrane-bound or soluble purified receptor are consistent, under the conditions used, with a single binding site of high affinity. Molecular cloning at present in progress should define the amino acid sequence and subunit composition of the human placental CGRP receptor, since at present the observed heterogeneity of CGRP-binding proteins may be interpreted in a number of ways, for instance: receptor heterogeneity, variable glycosylation of one of two subunits, or limited proteolysis of a single subunit during purification.     

Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy

A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences ofLabeo rohita, Cirrhina mrigala andCatla catla have been cloned, characterized and overexpressed inEscherichia coli. These sequences show 96–98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.     

Isolation and characterisation of cDNA clones for the A alpha- and gamma-chains of human fibrinogen.

cDNA clones coding for the A alpha- and gamma-chains of human fibrinogen have been isolated from an adult liver cDNA library. Clones were identified by hybridisation with mixtures of synthetic oligonucleotides 17 bases long, predicted using amino acid sequence data for each chain. The cDNA insert sizes are 1,950bp for A alpha-fibrinogen and 950bp for gamma-fibrinogen. The clones do not show any cross-hybridisation. Each cDNA hybridises to a unique sequence in the human genome. In adult human liver, Northern blots give an estimated messenger RNA size of 2.6kb for A alpha-fibrinogen and 1.8kb for gamma-fibrinogen.     

Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE)

Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.     

Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy

A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences ofLabeo rohita, Cirrhina mrigala andCatla catla have been cloned, characterized and overexpressed inEscherichia coli. These sequences show 96–98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.     

Isolation and characterisation of a cDNA encoding the precursor for human apolipoprotein AII

cDNA clones encoding human apolipoprotein AII have been isolated from an adult liver cDNA library. Apo AII mRNA was shown to be approximately 600 bases in length by RNA blot hybridisation. The intracellular precursor of apo AII was inferred from the cDNA sequence to be a 100 amino acid polypeptide consisting of the 77 residue mature protein and an additional 23 amino terminal residues. The amino terminal extension, divisible into an 18 residue signal peptide and a 5 residue propeptide, is separated from the first amino acid of mature apo AII by dibasic residues. The 5' untranslated region of the message is 61 bases in length and the 3' untranslated region 113 bases. A polyadenylation signal is situated 14 bases 3' of the poly(A) tail.     

Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines.

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA lambda gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.     

Isolation and characterisation of a cDNA clone for human apolipoprotein CI and assignment of the gene to chromosome 19

Summary We have synthesised a mixed oligonucleotide 17 bases long and used it to isolate cDNA clones for apolipoprotein CI (apo CI) from an adult liver cDNA library. The partial sequence of one of these clones confirms its identity. We have used this probe and Southern blotting techniques to identify the human apo CI gene in DNA from a series of rodent x human somatic cell hybrids. Our Results provide evidence for the assignment of this gene to human chromosome 19.