PMID- 2291473
OWN - NLM
STAT- MEDLINE
DCOM- 19910402
LR  - 20190627
IS  - 0003-2697 (Print)
IS  - 0003-2697 (Linking)
VI  - 190
IP  - 2
DP  - 1990 Nov 1
TI  - Amplification, detection, and automated sequencing of gibbon interleukin-2 mRNA 
      by Thermus aquaticus DNA polymerase reverse transcription and polymerase chain 
      reaction.
PG  - 292-6
AB  - Reverse transcription-polymerase chain reaction (RT-PCR) is a gene expression 
      assay by which messenger RNA (mRNA) production can be measured. This technique 
      involves three steps: isolation of RNA from cells or tissues, the creation of a 
      DNA copy of the desired message (cDNA) by viral reverse transcriptase enzymes 
      (RT), and amplification of this DNA segment by the polymerase chain reaction 
      (PCR) for subsequent quantitation and analysis. Here we describe a one-enzyme, 
      one-step method combining the RT and PCR steps of conventional RT-PCR by 
      exploiting the recently documented RT properties of Taq polymerase, the 
      thermostable enzyme used for PCR amplification of DNA. RNA was extracted from 
      gibbon T-cells (MLA144), reverse transcribed and amplified with oligonucleotide 
      primers (specific for the 5' portion of a spliced interleukin-2 (IL-2) messenger 
      RNA) by Taq polymerase. A discrete fragment of correct length for IL-2 cDNA was 
      detected. Experiments showed that this product was RNA-dependent and specific for 
      IL-2. This fragment was sequenced by automation employing a biotin 
      primer-streptavidin magnetic bead protocol which confirmed its origin as 
      processed IL-2 mRNA. The modification of the RT-PCR procedure using a 
      thermostable enzyme speeds up reaction time and increases stringency. This method 
      should make the diagnostic screening of cells for gene expression more efficient 
      and practical.
FAU - Shaffer, A L
AU  - Shaffer AL
AD  - Naval Medical Research Institute, Immunobiology, and Transplantation Department, 
      Bethesda, Maryland 20814-5055.
FAU - Wojnar, W
AU  - Wojnar W
FAU - Nelson, W
AU  - Nelson W
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (DNA, Bacterial)
RN  - 0 (Interleukin-2)
RN  - 0 (RNA, Messenger)
RN  - EC 2.7.7.- (Taq Polymerase)
RN  - EC 2.7.7.7 (DNA-Directed DNA Polymerase)
SB  - IM
MH  - Animals
MH  - Autoanalysis
MH  - Base Sequence
MH  - DNA, Bacterial/*chemistry
MH  - DNA-Directed DNA Polymerase
MH  - Gene Expression
MH  - Hylobates
MH  - Interleukin-2/*genetics
MH  - Lymphoma/genetics
MH  - Molecular Sequence Data
MH  - *Polymerase Chain Reaction
MH  - RNA Splicing
MH  - RNA, Messenger/*biosynthesis
MH  - Taq Polymerase
MH  - Thermus/*genetics
MH  - Transcription, Genetic
EDAT- 1990/11/01 00:00
MHDA- 1990/11/01 00:01
CRDT- 1990/11/01 00:00
PHST- 1990/11/01 00:00 [pubmed]
PHST- 1990/11/01 00:01 [medline]
PHST- 1990/11/01 00:00 [entrez]
AID - 0003-2697(90)90196-G [pii]
AID - 10.1016/0003-2697(90)90196-g [doi]
PST - ppublish
SO  - Anal Biochem. 1990 Nov 1;190(2):292-6. doi: 10.1016/0003-2697(90)90196-g.