PMID- 2472758
OWN - NLM
STAT- MEDLINE
DCOM- 19890810
LR  - 20190627
IS  - 0003-2697 (Print)
IS  - 0003-2697 (Linking)
VI  - 177
IP  - 1
DP  - 1989 Feb 15
TI  - Rapid amplification of complementary DNA from small amounts of unfractionated 
      RNA.
PG  - 7-10
AB  - We have combined, in a rapid and straightforward manner, the synthesis and 
      subsequent amplification of individual cDNA sequences from microgram quantities 
      of unfractionated total RNA. Taq1 polymerase, a thermostable DNA polymerase, and 
      Moloney murine leukemia virus (MMLV) reverse transcriptase share similar buffer 
      conditions; these reactions can be performed sequentially, in a single tube, 
      without the need for purification or changes of buffer after the synthesis of 
      cDNA. In this way, nonspecific losses of material are minimized and the required 
      number of cells is reduced. Cell numbers, particularly from human tissues, can be 
      limiting; the requirement for only small amounts of unfractionated RNA makes 
      possible the isolation and characterization of cDNAs from biological materials 
      available in limited quantities. As a demonstration system, we report the rapid 
      synthesis and amplification of cDNA sequences corresponding to the first exon of 
      human immunoglobulin E (IgE). MMLV reverse transcriptase is used with specific 
      (i.e., IgE) or generic (i.e., oligo-[dT(12-18)]) oligomers to prime first strand 
      cDNA synthesis from unfractionated RNA isolated from a human myeloma line, U-266. 
      The necessary primers, deoxynucleotides and Taq1 polymerase, required for second 
      strand cDNA synthesis and the subsequent logarithmic amplification process, are 
      then added to the reaction mixture. This technique provides a useful means of 
      characterizing expressed and processed gene transcripts.
FAU - Doherty, P J
AU  - Doherty PJ
AD  - Department of Immunology and Rheumatology, Hospital for Sick Children, Toronto, 
      Ontario, Canada.
FAU - Huesca-Contreras, M
AU  - Huesca-Contreras M
FAU - Dosch, H M
AU  - Dosch HM
FAU - Pan, S
AU  - Pan S
LA  - eng
GR  - GM-38420/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 63231-63-0 (RNA)
RN  - 9007-49-2 (DNA)
RN  - EC 2.7.7.- (Taq Polymerase)
RN  - EC 2.7.7.49 (RNA-Directed DNA Polymerase)
RN  - EC 2.7.7.7 (DNA-Directed DNA Polymerase)
SB  - IM
MH  - DNA/*genetics
MH  - DNA-Directed DNA Polymerase
MH  - *Gene Amplification
MH  - Humans
MH  - RNA/genetics
MH  - RNA-Directed DNA Polymerase
MH  - Taq Polymerase
EDAT- 1989/02/15 00:00
MHDA- 1989/02/15 00:01
CRDT- 1989/02/15 00:00
PHST- 1989/02/15 00:00 [pubmed]
PHST- 1989/02/15 00:01 [medline]
PHST- 1989/02/15 00:00 [entrez]
AID - 0003-2697(89)90003-1 [pii]
AID - 10.1016/0003-2697(89)90003-1 [doi]
PST - ppublish
SO  - Anal Biochem. 1989 Feb 15;177(1):7-10. doi: 10.1016/0003-2697(89)90003-1.