From 9996bc6cd1b8a9622bd976f3a81091f07ebc0982 Mon Sep 17 00:00:00 2001 From: Charles Date: Fri, 14 Feb 2020 12:41:39 +0900 Subject: [PATCH] =?utf8?q?Caf=C3=A9?= MIME-Version: 1.0 Content-Type: text/plain; charset=utf8 Content-Transfer-Encoding: 8bit --- biblio/Chenchick_1998.mdwn | 28 ++++++++++++++++++++++++++++ tags/template_switching.mdwn | 6 ++++++ 2 files changed, 34 insertions(+) create mode 100644 biblio/Chenchick_1998.mdwn diff --git a/biblio/Chenchick_1998.mdwn b/biblio/Chenchick_1998.mdwn new file mode 100644 index 00000000..50f0ed52 --- /dev/null +++ b/biblio/Chenchick_1998.mdwn @@ -0,0 +1,28 @@ +[[!meta title="Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR."]] +[[!tag template_switching]] + +Alex Chenchick, York Y. Shu, Luda Diatchenko, Roger Li, Jason Hill and Paul D. Siebert. +(Gene Cloning and Analysis Group, CLONETECH Laboratories, Pao Alto, CA, USA). + +In: Gene Cloning and Analysis by RT-PCR. Edited by Paul Siebert and James Larrick. 1998 + +Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR. + +Reaction mixture: 1 µM RTP; 1 µM TSO; 50–1000 ng total RNA; 2 mM DTT, 1 mM dNTP, 200 U SSII in 10 µL. + + +DNA/RNA ends tested: HO-G, Cap-G, HO-A, Cap-A, HO-C, Cap-C, HO-T + +TSOs tested: rG, rGrG, rGrGrG, rGrGrGrGrG, rUrUrU, GGG, rGrGrG in all-r oligo. + +With the wild-type MMLVm the HO-G DNA/RNA duplex is tailed with 1~5 extra +nucleotides (Fig 2). Using radiolabelled nucleotides suggests that they are +mostly Cs. "Not shown" experiments suggest that the presence of a cap "does +not significantly influence the preference of addition of these non-templated +nucleotides". The consensus tail is AACCC. SSII (RNAseH-) has a lower +efficiency for adding nucleotides, compared with wild-type MMLV. + +Template-switching is more efficient with at least 2 rG. dG is notably less +efficient and rU has no visible efficiency (Figure 2). + +In 2 % of the cDNAs, RT was primed by the TSO. diff --git a/tags/template_switching.mdwn b/tags/template_switching.mdwn index c4165c23..3945efb2 100644 --- a/tags/template_switching.mdwn +++ b/tags/template_switching.mdwn @@ -6,6 +6,12 @@ with the CapFinder PCR cDNA library construction kit.” Zhu, Y., A. Chenchik and P.D. Siebert. CLONTECHniques 1 1:12-13. Could not find the PDF. + - 1998: “Generation and use of high-quality cDNA from small amounts of total + RNA by SMART PCR” [[Chenchick and coll., 1998|biblio/Chenchick_1998]]. + Oligo-dT-primed total RNA is template-switched with rGrGrG DNA/RNA hybrids. + SMART means “Switch Mechanism At the 5′ end of RNA Templates”. Is that + the primary paper for SMART ? + - In the "_CapSelect_" method, [[Schmidt and Mueller, 1999|biblio/10518626]] stimulate template switching with manganese (see below), tail the first-strand cDNAs with dA, and add 5′ linkers with T4 DNA ligase and duplex adapters -- 2.47.3