Inverse PCR

For some useful biocatalysts, bacteria may not be the only source, and retrieving the gene from eukaryotic organisms might be necessary. 

To secure a gene from a eukaryotic genome, the most efficient method among several is to obtain cDNA (complementary DNA) from mRNA via reverse transcriptase. Use of cDNA avoids dealing with the problem of introns, where a complete gene can stretch over thousands of base pairs but is divided into several small exons, alternating with introns of sometimes remarkable size [several thousand base pairs (kB) for one intron]. As, however, only a few eukaryotic genes are fully sequenced and deposited in one of the databases, one has to rely on ESTs (expressed 

To obtain a template for the RACE, RNA isolation is necessary to rewrite it into cDNA. Precautions have to be taken, as RNA is a considerably more unstable molecule than DNA and, to add to the situation, its degrading enzymes are among the most stable known. Nowadays, most suppliers provide RNAse-free buffers and water, so DEPC (diethylpyrocarbonate) treatment to destroy RNAse activity is necessary only for home-made buffers. Kits for RNA preparation, either for total RNA or for mRNA isolation, do not require any DEPC treatment 

After obtaining pure total RNA from eukaryotic cells, the RNA needs to be rewritten into cDNA to serve as a template in PCR, as RNA cannot be amplified by PCR. The task of rewriting is accomplished with reverse transcriptase (RT), a viral enzyme used by retroviruses (whose name stems from harboring this enzyme and the ability to rewrite RNA into cDNA). The group of retroviruses has such members as the AIDS virus, Avian myeloblastosis virus, Murine leukemia virus (Frohmann, 1988 Kawasaki, 1988), and Adenovirus. Commonly employed reverse transcriptases stem from either the avian virus (AMV-RT) or the murine leukemia virus (MMLV, used in the Clontech RT-kit). 

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Forty-eight variants of p53 were generated by inverse PCR using the wild-type p53 expression vector, pQE-80L H6-BCCP-p53, as template. Phosphory-lated forward primers bore the sequence variation at the 5 -terminus followed by 20-24 nucleotides ofp53 sequence. Unphosphorylated reverse primers were... 

Inverse PCR can, in theory, be used to estimate the number of insertions of T-DNA into a plant genome. A recent report by Does et al. (1991) described the use of this approach to detect the presence and number of T-... 

Recovery of DNA Sequences Flanking P-element Insertions Inverse PCR and Plasmid Rescue ... 

The inverse PCR protocol given below was developed to isolate genomic DNA sequences immediately adjacent to the insertion of large numbers of P-elements as part of the gene-disruption project of the BDGP It has been successfully applied to various P-ele-ments including the P PZ (Mlodzik and Hiromi 1992), P lacW (Bier et al 1989), and P EP (Rorth 1996) elements. Sequences and maps of these P-elements can be found on Flybase at ... 

Protocol 23.1 describes a standard fly miniprep that requires very few flies and produces high-quality DNA. This protocol can also be used to isolate RNA when RNase-ffee conditions are utilized an extra step must be taken to rid the sample of genomic DNA (e.g., RNase-ffee DNase digestion). Genomic DNA prepared as in Protocol 23.1 is then digested with restriction enzymes (Protocol 23.2) and ligated (Protocol 23.3), and used for either plasmid rescue or inverse PCR as described below. 

Inverse PCR 5 (left) 5 (right) 5 (left) 3 (right) 5 (left) y (right)... 

Remove supernatant, wash the pellet with 70% ethanol, air-dry, and resuspend in 150 pi of sterile H O for inverse PCR and 10 pi of sterile H O for plasmid rescue. At this point, the ligated DNA can be stored at -20 C indefinitely. 

Where two primers are listed, the inverse PCR product can be sequenced from both ends by setting up sequencing reactions with the two different primers. 

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