Long-distance PCR.

Full-length cDNA synthesis for long-distance RT-PCR of large mRNA transcripts.

Reverse transcription of mRNA often leads to the synthesis of partial, non-full-length cDNAs. Methods to facilitate reverse transcription across RNA regions of secondary structure, as well as enzyme modifications to eliminate RNase H activities inherent to reverse transcriptase enzymes, have been previously reported. However, because all reverse transcriptases have high error rates of polymeriz...

Relative amplification efficiency of differently sized templates by long-distance PCR.

Multiplex co-amplification of 24 retinoblastoma gene exons after pre-amplification by long-distance PCR.

Polymerase chain reaction (PCR) amplification has become the method of choice for preparing the DNA template in mutation analysis from complex mixtures of DNA or RNA molecules. This strategy is optimal for small genes or genes with mutational hotspots. However, PCR-based mutation detection is neither labour nor cost effective when large or multiple genes, with many target fragments, are involve...

Long-distance PCR-based strategy for preparing knock-in vectors directly from ES cell genomic DNA.

be ligated to DNA fragments from any source, e.g., cDNAs obtained after subtractive hybridization or DNA fragments from S1 mapping experiments for analyzing exon-intron boundaries of genomic DNA. Due to the high ligation efficiency of dsPPL to the denatured ssDNA, the subsequent amplification is very efficient. It should be emphasized that PPL can be any palindromic structure that has three ran...

Long and accurate PCR.

INTRODUCTION The following protocol illustrates the rules of successful long PCR: No more than 1 ng of template DNA is used per microliter of PCR in a 100-μl reaction; approximately 0.1 μl of KlentaqLA (not plain Taq) is used per kilobase of target (for targets >10 kb, 1-1.3 μl of enzyme should be used); the Mg++ concentration is considered as the excess over the level of dNTPs; a final concent...