Template-switching polymerase chain reaction

Template-switching polymerase chain reaction (TS-PCR) is a method of reverse transcription and polymerase chain reaction (PCR) amplification that relies on a natural PCR primer sequence at the polyadenylation site, also known as the poly(A) tail, and adds a second primer through the activity of murine leukemia virus reverse transcriptase.[1] This permits reading full cDNA sequences and can deliver high yield from single sources, even single cells that contain 10 to 30 picograms of mRNA, with relatively low levels (3-5%) of contaminating rRNA sequence. This technique is often employed in whole transcriptome shotgun sequencing. It is marketed by Clontech as Switching Mechanism At the 5' end of RNA Template (SMART)[2][3] as well as by Diagenode as Capture and Amplification by Tailing and Switching  (CATS).

## Drop-Seq

By using syringe pumps to transmit a steady rate of isolated cells and uniquely oligonucleotide-barcoded beads, it is possible to isolate individual cells and beads together in droplets of lysis buffer, where the polyadenylation site binds to a primer containing a unique, identifying sequence.[4] This primer also contains a common sequence upstream of the identifier, so that after it is extended by reverse transcription, subsequent rounds of PCR will incorporate the tag, which permits each isolated cDNA that is sequenced to be tracked back to a specific originating bead. This permits the relative levels of transcripts in many individual cells to be analyzed simultaneously, creating a rational basis for the classification of these cells into particular cell types,[5] or permitting the logical inference of in situ hybridization data from embryos without actually performing the experiment.[6]

## References

1. ↑ L. Petalidis; S. Bhattacharyya; G. A. Morris; V. P. Collins; T. C. Freeman; P. A. Lyons (2003-11-15). "Global amplification of mRNA by template-switching PCR: linearity and application to microarray analysis". Nucleic Acids Research 31 (22): e142. doi:10.1093/nar/gng142. PMID 14602935.
2. ↑ "Transcriptome analysis from single cells, enabled by SMARTer technology". Clontech. http://www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=75905&minisite=10020&secItmId=20700.
3. ↑ "Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction". BioTechniques 30 (4): 892–7. April 2001. doi:10.2144/01304pf02. PMID 11314272.
4. ↑ Steve McCarroll. "McCarroll lab". Harvard. http://mccarrolllab.com/dropseq/. (includes updated protocol, publication link, software and datasets)
5. ↑ Emily Underwood (2015-08-07). "The brain's identity crisis". Science 349 (6248): 575–577. doi:10.1126/science.349.6248.575. PMID 26250665.
6. ↑ Rahul Satija; Jeffrey A Farrell; David Gennert; Alexander F Schier; Aviv Regev1 (2015). "Spatial reconstruction of single-cell gene expression data". Nature Biotechnology 33 (5): 495–502. doi:10.1038/nbt.3192. PMID 25867923.

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Polymerase chain reaction techniques

Procedure|

* Initialization
* cycle
* Denaturation
* Annealing
* elongation
* Final elongation
* Final hold

Polymerase|

* Klenow
* Taq
* Pfu

optimization
＆Variants|

* Quantitative polymerase chain reaction (qPCR)
* Reverse transcription polymerase chain reaction (RT-PCR)
* Inverse polymerase chain reaction
* Nested polymerase chain reaction
* Touchdown polymerase chain reaction
* Hot start PCR
* Overlap extension polymerase chain reaction
* Multiplex polymerase chain reaction
* Multiplex ligation-dependent probe amplification

history
＆people|

* Kary Mullis
* Kjell Kleppe [no; no]
* Alice Chien

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