Template switching polymerase chain reaction

From Wikipedia, the free encyclopedia
Jump to navigation Jump to search

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 and adds a second primer through the activity of murine leukemia virus reverse transcriptase.[1] This permits reading full cDNA sequence 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. Frequently the goal is some form of whole transcriptome shotgun sequencing. It is marketed by Clontech as Switching Mechanism At the 5' end of RNA Template (SMART).[2][3]. It is marketed by Diagenode as Capture and Amplification by Tailing and Switching (CATS).

Drop-Seq[edit]

By using syringe pumps to transmit a steady rate of isolated cells and uniquely barcoded beads, it is possible to isolate individual cells and beads together in droplets of lysis buffer, where the polyadenylation site binds to a bead-specific 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[edit]

  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. PMC 275579Freely accessible. PMID 14602935. 
  2. ^ "Transcriptome analysis from single cells, enabled by SMARTer technology". Clontech. 
  3. ^ Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD (April 2001). "Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction". Biotechniques. 30 (4): 892–7. PMID 11314272. 
  4. ^ Steve McCarroll. "McCarroll lab". Harvard.  (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: 495–502. doi:10.1038/nbt.3192.