T7 RNA POLYMERASE

Uses

• T7 RNA Polymerase can transcribe RNA from cloned DNA templates that are downstream from a T7 promoter. The synthesis can be performed with labeled NTPs to generate highly labeled RNA. Synthesized RNA can be used in many applications, including: RNA or DNA blotting techniques
• In situ hybridization
• RNase protection studies: Transcripts synthesized by the enzyme are used as precursor RNA for studies on RNA splicing and processing.
• Synthesis of capped RNA in vitro with addition of m7GpppG or m7GpppA in excess over GTP or ATP during the transcription reaction. The generated antisense RNA can be introduced into cells to suppress the expression of the corresponding genes.
• Microarray target synthesis

General Description

T7 RNA polymerase is commonly used to transcribe DNA which has been cloned into vectors which have two phage promoters in opposite orientation. RNA can be selectively synthesized from either strand of the insert DNA with different polymerases. Homogeneously labeled single-stranded RNA can be generated with this system. Transcripts can be non-radioactively labeled with biotin or DIG-11-UTP or radioactively labeled to high specific activity with [α-³²P] or [α-³⁵S]-labeled nucleotides.

Synthesis of hybridization probes: T7 RNA polymerase allows highly efficient production of homogeneously labeled RNA. This labeled RNA may be used as hybridization probes in Southern, northern, and dot blots, as well as in situ hybridizations.
Suitable labels:Transcripts can be nonradioactively labeled with biotin-16-UTP or DIG-11-UTP. They may also be radioactively labeled to high specific activity with [α-32P]- or [α-35S]-labeled nucleotides.