Enzymatic amplification of myosin heavy-chain mRNA sequences in vitro.
Identifieur interne : 001A10 ( Ncbi/Checkpoint ); précédent : 001A09; suivant : 001A11Enzymatic amplification of myosin heavy-chain mRNA sequences in vitro.
Auteurs : P. Harbarth [Allemagne] ; H P VosbergSource :
- DNA (Mary Ann Liebert, Inc.) [ 0198-0238 ] ; 1988.
Descripteurs français
- KwdFr :
- ARN messager (analyse), ARN messager (génétique), Amplification de gène, Animaux, Clonage moléculaire (), DNA restriction enzymes, Données de séquences moléculaires, Fragments peptidiques (génétique), Gènes, Humains, Hybridation d'acides nucléiques, Lapins, Muscles (métabolisme), Myocarde (métabolisme), Myosines (génétique), Sous-fragments de myosine, Spécificité d'espèce, Spécificité d'organe, Séquence nucléotidique.
- MESH :
- analyse : ARN messager.
- génétique : ARN messager, Fragments peptidiques, Myosines.
- métabolisme : Muscles, Myocarde.
- Amplification de gène, Animaux, Clonage moléculaire, DNA restriction enzymes, Données de séquences moléculaires, Gènes, Humains, Hybridation d'acides nucléiques, Lapins, Sous-fragments de myosine, Spécificité d'espèce, Spécificité d'organe, Séquence nucléotidique.
English descriptors
- KwdEn :
- Animals, Base Sequence, Cloning, Molecular (methods), DNA Restriction Enzymes, Gene Amplification, Genes, Humans, Molecular Sequence Data, Muscles (metabolism), Myocardium (metabolism), Myosin Subfragments, Myosins (genetics), Nucleic Acid Hybridization, Organ Specificity, Peptide Fragments (genetics), RNA, Messenger (analysis), RNA, Messenger (genetics), Rabbits, Species Specificity.
- MESH :
- chemical , analysis : RNA, Messenger.
- chemical , genetics : Myosins, Peptide Fragments, RNA, Messenger.
- chemical : DNA Restriction Enzymes, Myosin Subfragments.
- metabolism : Muscles, Myocardium.
- methods : Cloning, Molecular.
- Animals, Base Sequence, Gene Amplification, Genes, Humans, Molecular Sequence Data, Nucleic Acid Hybridization, Organ Specificity, Rabbits, Species Specificity.
Abstract
We have developed a procedure that detects the presence of mRNA coding for human beta-myosin heavy chain in small amounts of total, unfractionated RNA isolated from heart or skeletal muscle. The protocol is based on the enzymatic amplification in vitro of a selected 106-bp myosin isotype-specific subregion of this mRNA. The method, which is a modification of the so-called "polymerase chain reaction," requires two synthetic oligonucleotide primers (20-mers), reverse transcriptase, and DNA polymerase I (Klenow fragment). Two principle steps are involved: (i) the selected mRNA subregion is converted into a double-stranded cDNA, and (ii) this cDNA is amplified in 22 synthetic cycles. After gel electrophoresis and blotting the amplification product is identified by hybridization with a third oligonucleotide recognizing the region between the two primer annealing sites, and by restriction mapping. Only mRNA from muscle tissue promoted formation of the amplified 106-bp fragment. We estimate that less than 30,000 beta-myosin heavy-chain mRNA molecules are sufficient to produce a signal. The procedure is fast, specific, and very sensitive. It may be used in muscle gene expression studies with small numbers of cells or even in single muscle fibers.
DOI: 10.1089/dna.1988.7.297
PubMed: 2840250
Affiliations:
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Harbarth</name><affiliation wicri:level="3"><nlm:affiliation>Max-Planck-Institut für medizinische Forschung, Abteilung Molekulare Biologie, Heidelberg, FRG.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Max-Planck-Institut für medizinische Forschung, Abteilung Molekulare Biologie, Heidelberg</wicri:regionArea><placeName><region type="land" nuts="1">Bade-Wurtemberg</region><region type="district" nuts="2">District de Karlsruhe</region><settlement type="city">Heidelberg</settlement></placeName></affiliation></author><author><name sortKey="Vosberg, H P" sort="Vosberg, H P" uniqKey="Vosberg H" first="H P" last="Vosberg">H P Vosberg</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="1988">1988</date><idno type="RBID">pubmed:2840250</idno><idno type="pmid">2840250</idno><idno type="doi">10.1089/dna.1988.7.297</idno><idno type="wicri:Area/PubMed/Corpus">002A60</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002A60</idno><idno type="wicri:Area/PubMed/Curation">002A60</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">002A60</idno><idno type="wicri:Area/PubMed/Checkpoint">002903</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">002903</idno><idno type="wicri:Area/Ncbi/Merge">001A10</idno><idno type="wicri:Area/Ncbi/Curation">001A10</idno><idno type="wicri:Area/Ncbi/Checkpoint">001A10</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Enzymatic amplification of myosin heavy-chain mRNA sequences in vitro.</title><author><name sortKey="Harbarth, P" sort="Harbarth, P" uniqKey="Harbarth P" first="P" last="Harbarth">P. Harbarth</name><affiliation wicri:level="3"><nlm:affiliation>Max-Planck-Institut für medizinische Forschung, Abteilung Molekulare Biologie, Heidelberg, FRG.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Max-Planck-Institut für medizinische Forschung, Abteilung Molekulare Biologie, Heidelberg</wicri:regionArea><placeName><region type="land" nuts="1">Bade-Wurtemberg</region><region type="district" nuts="2">District de Karlsruhe</region><settlement type="city">Heidelberg</settlement></placeName></affiliation></author><author><name sortKey="Vosberg, H P" sort="Vosberg, H P" uniqKey="Vosberg H" first="H P" last="Vosberg">H P Vosberg</name></author></analytic><series><title level="j">DNA (Mary Ann Liebert, Inc.)</title><idno type="ISSN">0198-0238</idno><imprint><date when="1988" type="published">1988</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Cloning, Molecular (methods)</term><term>DNA Restriction Enzymes</term><term>Gene Amplification</term><term>Genes</term><term>Humans</term><term>Molecular Sequence Data</term><term>Muscles (metabolism)</term><term>Myocardium (metabolism)</term><term>Myosin Subfragments</term><term>Myosins (genetics)</term><term>Nucleic Acid Hybridization</term><term>Organ Specificity</term><term>Peptide Fragments (genetics)</term><term>RNA, Messenger (analysis)</term><term>RNA, Messenger (genetics)</term><term>Rabbits</term><term>Species Specificity</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN messager (analyse)</term><term>ARN messager (génétique)</term><term>Amplification de gène</term><term>Animaux</term><term>Clonage moléculaire ()</term><term>DNA restriction enzymes</term><term>Données de séquences moléculaires</term><term>Fragments peptidiques (génétique)</term><term>Gènes</term><term>Humains</term><term>Hybridation d'acides nucléiques</term><term>Lapins</term><term>Muscles (métabolisme)</term><term>Myocarde (métabolisme)</term><term>Myosines (génétique)</term><term>Sous-fragments de myosine</term><term>Spécificité d'espèce</term><term>Spécificité d'organe</term><term>Séquence nucléotidique</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>RNA, Messenger</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Myosins</term><term>Peptide Fragments</term><term>RNA, Messenger</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>DNA Restriction Enzymes</term><term>Myosin Subfragments</term></keywords><keywords scheme="MESH" qualifier="analyse" xml:lang="fr"><term>ARN messager</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ARN messager</term><term>Fragments peptidiques</term><term>Myosines</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Muscles</term><term>Myocardium</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Cloning, Molecular</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Muscles</term><term>Myocarde</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Base Sequence</term><term>Gene Amplification</term><term>Genes</term><term>Humans</term><term>Molecular Sequence Data</term><term>Nucleic Acid Hybridization</term><term>Organ Specificity</term><term>Rabbits</term><term>Species Specificity</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Amplification de gène</term><term>Animaux</term><term>Clonage moléculaire</term><term>DNA restriction enzymes</term><term>Données de séquences moléculaires</term><term>Gènes</term><term>Humains</term><term>Hybridation d'acides nucléiques</term><term>Lapins</term><term>Sous-fragments de myosine</term><term>Spécificité d'espèce</term><term>Spécificité d'organe</term><term>Séquence nucléotidique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">We have developed a procedure that detects the presence of mRNA coding for human beta-myosin heavy chain in small amounts of total, unfractionated RNA isolated from heart or skeletal muscle. The protocol is based on the enzymatic amplification in vitro of a selected 106-bp myosin isotype-specific subregion of this mRNA. The method, which is a modification of the so-called "polymerase chain reaction," requires two synthetic oligonucleotide primers (20-mers), reverse transcriptase, and DNA polymerase I (Klenow fragment). Two principle steps are involved: (i) the selected mRNA subregion is converted into a double-stranded cDNA, and (ii) this cDNA is amplified in 22 synthetic cycles. After gel electrophoresis and blotting the amplification product is identified by hybridization with a third oligonucleotide recognizing the region between the two primer annealing sites, and by restriction mapping. Only mRNA from muscle tissue promoted formation of the amplified 106-bp fragment. We estimate that less than 30,000 beta-myosin heavy-chain mRNA molecules are sufficient to produce a signal. The procedure is fast, specific, and very sensitive. It may be used in muscle gene expression studies with small numbers of cells or even in single muscle fibers.</div></front></TEI><affiliations><list><country><li>Allemagne</li></country><region><li>Bade-Wurtemberg</li><li>District de Karlsruhe</li></region><settlement><li>Heidelberg</li></settlement></list><tree><noCountry><name sortKey="Vosberg, H P" sort="Vosberg, H P" uniqKey="Vosberg H" first="H P" last="Vosberg">H P Vosberg</name></noCountry><country name="Allemagne"><region name="Bade-Wurtemberg"><name sortKey="Harbarth, P" sort="Harbarth, P" uniqKey="Harbarth P" first="P" last="Harbarth">P. Harbarth</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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