Café

diff --git a/biblio/Chenchick_1998.mdwn b/biblio/Chenchick_1998.mdwn
new file mode 100644 (file)
index 0000000..50f0ed5
--- /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 c4165c23089fd7ce14b142af5dcf396d4551d638..3945efb2e05380da6fd9caf1b7acf754b465081f 100644 (file)
--- 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