Bacillus stearothermophilus reverse transcription compositions and kits

#The present invention relates to reverse transcription of RNA, and in particular to thermostable DNA polymerases that have reverse transcriptase activity.

#Many ribonucleic acid (RNA) molecules contain secondary structure that results from hybridization between complementary regions within the RNA molecule. A variety of secondary structures can be formed, including hairpins and cruciforms. RNA molecules containing secondary structure are often difficult to reverse transcribe because polymerases cannot readily process through the secondary structure.

#Because of the difficulty of reverse transcribing RNA molecules with secondary structure, many techniques dependent upon reverse transcription yield anomalous results. For example, RNA molecules with secondary structure may be poorly represented in cDNA libraries. Populations of RNA with secondary structure may also yield cDNA libraries with a short insert size. Furthermore, RNA molecules containing secondary structure may be difficult to detect in assays such as reverse transcription-polymerase chain reaction (RT-PCR).

#FIG. 1 provides the single letter code nucleotide for the DNA sequence encoding full-length Tvu DNA polymerase (SEQ ID NO: 1).

#FIG. 2 provides the predicted amino acid sequence of full-length Tvu DNA polymerase (SEQ ID NO: 2).

#FIG. 3 provides the DNA sequence encoding the 5′ to 3′ exonuclease deletion mutant form of Tvu DNA polymerase called M285. This DNA sequence encodes the enzyme beginning at the nucleotides encoding the methionine amino acid at position 285 of wild type Tvu DNA polymerase and ending at the termination codon of the wild type enzyme (SEQ ID NO: 3).

#To facilitate understanding of the invention, a number of terms are defined below.

#The term “gene” as used herein, refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or protein precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence, as long as the desired protein activity is retained.

#“Nucleoside”, as used herein, refers to a compound consisting of a purine [guanine (G) or adenine (A)] or pyrimidine [thymine (T), uridine (U), or cytidine (C)] base covalently linked to a pentose, whereas “nucleotide” refers to a nucleoside phosphorylated at one of its pentose hydroxyl groups.