It allows the synthesis of cDNA fragments of up to 9 kbp. and include modified bases. It is active within 55°C – 60°C, allows complex RNA templates to be used and provides reaction specificity. It has RNase H activity (suitable for analyzing changes in gene expression). It provides a high yield of cDNA, the reaction yield is at least 100 ng of the first cDNA strand at 100 u. RNAscribe RT per 1 mcg of RNA. The fast reaction speed allows the synthesis to be carried out in as little as 15 minutes. The enzyme is resistant to incubation at room temperature for up to 15 days.
Areas of use
- cDNA synthesis for cloning
- Classic PCR with “hot” start
- DNA labelling
- Preparation of labelled cDNA probes for microarrays
reverse transcriptase also called RNA-directed DNA polymerase, is an enzyme encoded from the genetic material of retroviruses that catalyzes the transcription of retrovirus RNA (ribonucleic acid) into DNA (deoxyribonucleic acid). This catalyzed transcription is the reverse process of normal cellular transcription of DNA into RNA, hence the names reverse transcriptase and retrovirus.
Reverse transcriptase is central to the infectious nature of retroviruses, several of which cause disease in humans, including human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), and T-cell lymphotropic virus. human I (HTLV-I), which causes leukaemia. Reverse transcriptase is also a critical component of a laboratory technique known as reverse transcription-polymerase chain reaction (RT-PCR), a powerful tool used in the research and diagnosis of diseases such as cancer.
Retroviruses consist of an RNA genome contained within a protein coat that is enclosed in a lipid envelope. The retrovirus genome is typically composed of three genes: the group-specific antigen gene (gag), the polymerase gene (pol), and the envelope gene (env). The pol gene encodes the three enzymes (protease, reverse transcriptase, and integrase) that catalyze the steps of retroviral infection.
Once a retrovirus is inside a host cell (a protease-mediated process), it takes over the host’s genetic transcription machinery to build a DNA provirus. This process, the conversion of retroviral RNA to proviral DNA, is catalyzed by reverse transcriptase and is necessary for the insertion of proviral DNA into host DNA, a step initiated by the enzyme integrase.
Transcriber Reverse transcriptase is designed to transcribe RNA (mRNA, total RNA, viral RNA, and in vitro transcribed RNA) from a variety of sources, using conventional thermal cyclers and real-time PCR instruments (eg, LightCycler® instruments) for the following applications:
- Synthesis of first-strand cDNA for use in subsequent amplification reactions.
- RT-PCR of GC-rich RNA templates
- Cy3, Cy5, DIG, biotin, and aminoallyl labelling during cDNA synthesis
- Recovery and cloning of the 5 ′ and 3 ′ ends of mRNA by RACE
- Generation of cDNA libraries with large inserts.
- Dideoxy DNA sequencing
- RNA sequencing
- Marking the 3 ‘end of DNA fragments
- Generation of single-stranded probes for genomic fingerprints
- In the reverse transcription of human papillomavirus E6 RNA, cortical and striatal tissues, muscle biopsies from
- Becker muscular dystrophy specimens, and human brain microvascular endothelial cell and oocyte miRNA stem-loop (HCMC)
Biochemical / Physiological Actions
The reverse transcriptase of human immunodeficiency virus type 1 (HIV-1 RT) is essential for the catalytic conversion of single-stranded viral RNA into linear double-stranded DNA that is integrated into host cell chromosomes.
Features and Benefits
- Achieve high sensitivity in two-step RT-PCR.
Reverse Transcriptase Transcriptor is used in conventional thermal cyclers and real-time PCR instruments (eg LightCycler® instruments).
- Get more complete transcripts, up to 14 kb.
Large insert cDNA libraries can be generated.
- Transcribe difficult templates in reverse.
The enzyme works well at elevated temperatures, thereby overcoming the secondary structure of RNA (eg, GC-rich RNA templates) and providing optimal reaction conditions.
- Efficiently label the cDNA.
Nucleotides labelled with Cy3, Cy5, DIG, biotin, or aminoallyl are incorporated during cDNA synthesis.
- Transcriber Reverse Transcriptase, in storage buffer
- Transcriptional Buffer RT, 5x concentrated