Rapid Amplification Of cDNA Ends Protocol Research Articles

Next-generation sequencing-based 5′ rapid amplification of cDNA ends for alternative promoters

Mammalian genomes contain many unknown alternative first exons and promoters. Thus, we have modified the existing 5′RACE (5′ rapid amplification of cDNA ends) approach into a next-generation sequencing (NGS)-based new protocol that can identify these alternative promoters. This protocol has incorporated two main ideas: (i) 5′RACE starting from the known second exons of genes and (ii) NGS-based sequencing of the subsequent cDNA products. This protocol also provides a bioinformatics strategy that processes the sequence reads from NGS runs. This protocol has successfully identified several alternative promoters for an imprinted gene, PEG3. Overall, this NGS-based 5′RACE protocol is a sensitive and reliable method for detecting low-abundant transcripts and promoters.

Rapid Amplification of cDNA Ends for RNA Transcript Sequencing in Staphylococcus.

Rapid amplification of cDNA ends (RACE) is a technique that was developed to swiftly and efficiently amplify full-length RNA molecules in which the terminal ends have not been characterized. Current usage of this procedure has been more focused on sequencing and characterizing RNA 5' and 3' untranslated regions. Herein is described an adapted RACE protocol to amplify bacterial RNA transcripts.

RecA-mediated multistrand formation for cloning PCR products into vectors: Simplified process for 5′-rapid amplification of cDNA ends

I have developed a novel rapid amplification of cDNA ends (RACE) technology that uses multistranded DNA formation mediated by the RecA protein. Multistranded DNA can readily be formed at the terminus of double-stranded DNA by a complementary single-stranded DNA in the presence of RecA and exonuclease I. The possibility of applying this finding to the direct cloning of a 5′-RACE product onto a cDNA fragment, which does not require the use of restriction endonucleases, was explored. The results show that the terminal multistranded structure formed by the RecA-mediated reaction can be applied to RACE systems. Modifications to the RACE protocol to improve the effectiveness of the technique are also suggested.

Guinea pig 11β-hydroxysteroid dehydrogenase type 1: primary structure and catalytic properties☆

The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme is responsible for the interconversion of glucocorticoids and their inactive metabolites, and thus modulates the intracellular level of bioactive glucocorticoids. The present study was designed to clone and characterize 11β-HSD1 in the guinea pig, a laboratory animal known for resistance to glucocorticoids. The cDNA encoding guinea pig 11β-HSD1 was cloned by a modified 3′-RACE (rapid amplification of cDNA ends) protocol using the hepatic RNA as template. The cloned cDNA encodes a protein of 300 amino acids that shares 71 to 74% sequence identity with other known mammalian 11β-HSD1 proteins. Sequence comparison analysis revealed that the deduced guinea pig 11β-HSD1 was longer, by eight amino acids at the C terminus, than those of other mammals. Moreover, one of the two absolutely conserved consensus sites for N-glycosylation was absent. To examine the functional significance of these structural changes, we also characterized 11β-HSD1 activity in the hepatic microsomes. Although the guinea pig hepatic enzyme was NADP(H)-dependent and reversible, it displayed equal affinity for cortisol and cortisone (apparent K m for both substrates was 3 μM). This is in marked contrast to 11β-HSD1 in other mammals whose affinity for cortisone is approximately 10 times higher than that for cortisol (apparent K m of 0.3 vs. 3.0 μM). The apparent lower affinity of the guinea pig enzyme for cortisone would suggest that the intracellular bioformation of cortisol from circulating cortisone may be less efficient in this species. Northern blot analysis and RT-PCR revealed that the mRNA for 11β-HSD1 was widely expressed in the adult guinea pig but at low amounts. In conclusion, the present study has identified distinct features in the deduced primary structure and catalytic function of 11β-HSD1 in the guinea pig. Thus, the guinea pig provides a useful model in which the structural determinants of catalytic function of 11β-HSD1 may be studied.

On beyond classic RACE (rapid amplification of cDNA ends).

Department of Pharmacology, University Medical Center at Stony Brook, Stony Brook, New York 11794-8651 Most attempts to identify and isolate a novel cDNA result in the acquisition of clones that represent only a part of the mRNA's complete sequence. The missing sequence (cDNA ends) can be cloned by PCR, using a technique variously called rapid amplification of cDNA ends (RACE), (1) anchored PCR,(2) or one-sided PCR. (3) Since the initial reports of this technique, many laboratories have developed significant improvements on the basic approach. (4-16) I will describe here the most recent hybrid version of the relatively simple classic RACE, and a more powerful but technically more challenging new RACE protocol that is adapted from the work of a number of laboratories. ~ 24) Commercial RACE kits that are convenient but not as powerful as the most recent versions of classic and new RACE are available from Bethesda Research Laboratories(~l) and Clontech.

24] Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: Thermal race

Publisher Summary The generation of a full-length complementary DNA (cDNA) can represent the most challenging part of a cloning project, particularly with respect to obtaining the 5' end of the cDNA. The initial step––reverse transcription of messenger RNA (mRNA)—is also the most critical one, and many variations on it is described to maximize the likelihood of obtaining fully extended cDNA. Thermal rapid amplification of cDNA ends (RACE) is a polymerase chain reaction (PCR) technique through which previously unobtained 3' and 5' ends of a cDNA can be amplified starting with the knowledge of a small stretch of sequence from within an internal region of the cDNA. This technique provides an alternative to constructing and screening conventional libraries to obtain the remainder of the sequence for a partially cloned cDNA. Partial cDNAs are generated using PCR to amplify the copies of the region between a single point in a mRNA transcript and its 3' or 5' end. To use the RACE protocol, a short, internal stretch of sequence must already be known from the mRNA of interest.

Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein.

We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA indicates that it encodes a protein of the 425 amino acids [tentatively named adipose differentiation-related protein (ADRP)] that does not have any similarity with sequences contained in the GenBank DNA and Protein Identification Resource protein data bases. Immunoblot of 1246 cell extracts with an antibody raised against the expressed ADRP shows that the 1246 cells contain a 50-kDa protein, the production of which increases as the cells differentiate. Localization of ADRP in 1246 cells indicates that ADRP is absent from nuclear and cytosolic fractions and is found as a membrane-associated protein. These results demonstrate that adipocyte differentiation is accompanied by early expression of a mRNA encoding a membrane-associated adipose differentiation related protein that is adipose tissue specific in vivo.

Isolation and expression of a murine purine phosphorylase-encoding cDNA and sequence similarity with the human message

To isolate murine purine nucleoside phosphorylase-encoding cDNA sequences ( PNP), a murine BALB/c liver cDNA library in λgt10 was screened for recombinants hybridizing to a human PNP cDNA probe. Two of three clones recovered included inserts large enough to contain the full-length coding sequence. Sequence analysis of the largest clone revealed an 867-nucleotide open reading frame encoding 289 amino acids with 84% residue identity to that encoded by human PNP and 351 bp of 3′-untranslated region. The 5′ end of the murine PNP message was specifically amplified by PCR using the RACE (rapid amplification of cDNA ends) protocol, revealing a 5′-untranslated region of 78 bp. Northern hybridization using the murine PNP cDNA sequence as a probe identified a message of approx. 1.6 kb in mouse NIH3T3 cells which was slightly smaller than the human message observed in HeLa cells. The cloned murine PNP cDNA coding sequence was inserted into a mammalian expression vector under transcriptional regulation of the Moloney murine leukemia virus long terminal repeat. Transfection of this plasmid into human 293 cells resulted in the expression of PNP activity which co-focused with murine PNP activity extracted from NIH3T3 cells, verifying that the isolated murine PNP cDNA clone encoded catalytically active PNP protein.