What is a template switch oligo?
Answer: The TSO (template switch oligo) is an oligo that hybridizes to untemplated C nucleotides added by the reverse transcriptase during reverse transcription. The TSO adds a common 5′ sequence to full length cDNA that is used for downstream cDNA amplification.
Why is Oligo dT an effective primer for reverse transcriptase?
Oligo(dT) primers amplify only mRNAs containing a poly(A) tail, since that is where the primer binds to promote reverse transcription. Random primers amplify most RNA species, including degraded RNA and viral genomes.
What is the template for reverse transcriptase?
RNA template
Reverse transcriptases (RTs) use an RNA template and a short primer complementary to the 3′ end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR).
What is Oligo dT?
Oligo (dT)18 Primer is single-stranded sequence of deoxythymine (dT), used for priming reactions catalysed by reverse transcriptase. The transcript is primed in the poly(A) tail of mRNA molecules.
What is an anchor primer?
Anchor Primer™ is a consolidating micro acrylic water based insulator for internal and external use, and is composed of water soluble acrylic resin. This quartz primer permits the primer to be absorbed into the surface, creating a bond between the wall and the finish product you choose to apply.
What is smart seq?
Smart-Seq and Smart-Seq2: Switch Mechanism at the 5′ End of RNA Templates Smart-Seq was developed as a single-cell sequencing protocol with improved read coverage across transcripts. Complete coverage across the genome allows the detection of alternative transcript isoforms and SNPs.
What makes a good primer for reverse transcriptase?
Use random hexamer primers or gene-specific primers. Use reverse transcriptases which allow reverse transcription at higher temperatures, such as the Transcriptor First Strand cDNA Synthesis Kit.
Can reverse transcriptase use DNA as a template?
Reverse transcriptase is an enzyme found in retroviruses that converts the RNA genome carried in the retrovirus particle into double-stranded DNA. The single-stranded DNA is then used as a template for synthesizing double-stranded DNA (cDNA).
Why is cDNA used instead of mRNA?
When scientists use viral enzymes to make cDNA from RNA isolated from the cells and tissues that they are studying, it does not contain introns due to being spliced out in mRNA. cDNA also does not contain any other gDNA that does not directly code for a protein (referred to as non coding DNA).
Are primers oligos?
The term oligonucleotide is derived from the Greek “oligo,” which means few or small. Oligonucleotides made up of 2′-deoxyribonucleotides are the molecules used in polymerase chain reaction (PCR). These are referred to as primers and are used to massively amplify a small amount of DNA.
How long is oligo dT?
Specifications
| Product Type: | Primer |
|---|---|
| Primer Length: | 20 -mer |
| Primer Sequence: | 5’d PO4 [(T)20]3′ |
| Purification Method: | Gel-Purified, Desalted |
| Shipping Condition: | Approved for shipment on Wet or Dry Ice |
What is the difference between oligo and poly?
As nouns the difference between oligo and poly is that oligo is (biochemistry) an oligonucleotide while poly is dust.
How does reverse transcriptase switch to the TS oligo?
These bases function as a template switching (TS) oligo-anchoring site. Upon base pairing between the TS oligo and the appended deoxycytidine stretch, the reverse transcriptase “switches” template strands, from cellular RNA to the TS oligo, and continues replication to the 5′ end of the TS oligo.
How does reverse transcriptase switch template from RNA to RNA?
The reverse transcriptase (RT) in the Template Switching RT Enzyme Mix adds a few non-templated nucleotides after it reaches the 5′ end of the RNA template. These non-templated nucleotides can anneal to a template switching oligo (TSO) with a known sequence, prompting the reverse transcriptase to switch template from RNA to the TSO.
How are template switching oligos used for cDNA amplification?
Taken together, template switching (TS) oligos attach a universal primer-binding site through the activity of MMLV reverse transcriptase, enabling exclusive amplification of intact cDNA molecules. Template switching (TS) oligos containing 3′ rGs can be ordered from the Custom RNA Oligos ordering page on the IDT website.
Can a reverse transcriptase be used in cDNA construction?
Product spotlight: Conventional cDNA construction strategies usually result in an underrepresentation of the 5′ ends of cDNA. However, use of a template switching chimeric DNA:RNA oligo and MMLV reverse transcriptase can improve on this.