In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations
Ethanol fixation combined with microwave pretreatment allows rapid and simple detection of signals produced by cycling primed in situ (PRINS) amplification, which uses a single primer, and in situ polymerase chain reaction (ISPCR) in intact cells. After thermal cycling, signals remain as discrete su...
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| Veröffentlicht in: | Histochemistry and cell biology 1999-05, Vol.111 (5), p.411-416 |
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| creator | Paskins, L Brownie, J Bull, J |
| description | Ethanol fixation combined with microwave pretreatment allows rapid and simple detection of signals produced by cycling primed in situ (PRINS) amplification, which uses a single primer, and in situ polymerase chain reaction (ISPCR) in intact cells. After thermal cycling, signals remain as discrete subnuclear spots in the region of amplification and are clearly distinguishable from non-specific background labelling. These methods are applicable to routine blood smears, even after Giemsa staining or immunocytochemistry, and cellular morphology is retained. Chromosome enumeration by cycling PRINS is demonstrated using primers for repeat DNA sequences, whilst single copy sequence detection is demonstrated using bcl-2, CFTR and chromosome 21 specific primer pairs in ISPCR. We show that ethanol fixation supports efficient extension of cycling PRINS products to approximately 550 bp using up to 70 rounds of thermal cycling. |
| doi_str_mv | 10.1007/s004180050375 |
| format | Article |
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After thermal cycling, signals remain as discrete subnuclear spots in the region of amplification and are clearly distinguishable from non-specific background labelling. These methods are applicable to routine blood smears, even after Giemsa staining or immunocytochemistry, and cellular morphology is retained. Chromosome enumeration by cycling PRINS is demonstrated using primers for repeat DNA sequences, whilst single copy sequence detection is demonstrated using bcl-2, CFTR and chromosome 21 specific primer pairs in ISPCR. We show that ethanol fixation supports efficient extension of cycling PRINS products to approximately 550 bp using up to 70 rounds of thermal cycling.</description><identifier>ISSN: 0948-6143</identifier><identifier>EISSN: 1432-119X</identifier><identifier>DOI: 10.1007/s004180050375</identifier><identifier>PMID: 10403121</identifier><language>eng</language><publisher>Germany: Springer Nature B.V</publisher><subject>Adaptation ; Azure Stains ; Bcl-2 protein ; Cell Line ; Chromosome 21 ; Chromosomes, Human, Pair 21 ; Color ; Enumeration ; Ethanol ; Fixatives ; Gene Dosage ; Humans ; Immunocytochemistry ; Labeling ; Microwaves ; Nucleotide sequence ; Polymerase chain reaction ; Primed In Situ Labeling - methods ; Staining and Labeling - methods ; Thermal cycling ; Tissue Fixation</subject><ispartof>Histochemistry and cell biology, 1999-05, Vol.111 (5), p.411-416</ispartof><rights>Springer-Verlag Berlin Heidelberg 1999.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c383t-20602065856ff1e7184436969523ab2b127dd6c78b2053bcdbd48258b03446d93</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10403121$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Paskins, L</creatorcontrib><creatorcontrib>Brownie, J</creatorcontrib><creatorcontrib>Bull, J</creatorcontrib><title>In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations</title><title>Histochemistry and cell biology</title><addtitle>Histochem Cell Biol</addtitle><description>Ethanol fixation combined with microwave pretreatment allows rapid and simple detection of signals produced by cycling primed in situ (PRINS) amplification, which uses a single primer, and in situ polymerase chain reaction (ISPCR) in intact cells. 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We show that ethanol fixation supports efficient extension of cycling PRINS products to approximately 550 bp using up to 70 rounds of thermal cycling.</description><subject>Adaptation</subject><subject>Azure Stains</subject><subject>Bcl-2 protein</subject><subject>Cell Line</subject><subject>Chromosome 21</subject><subject>Chromosomes, Human, Pair 21</subject><subject>Color</subject><subject>Enumeration</subject><subject>Ethanol</subject><subject>Fixatives</subject><subject>Gene Dosage</subject><subject>Humans</subject><subject>Immunocytochemistry</subject><subject>Labeling</subject><subject>Microwaves</subject><subject>Nucleotide sequence</subject><subject>Polymerase chain reaction</subject><subject>Primed In Situ Labeling - methods</subject><subject>Staining and Labeling - methods</subject><subject>Thermal cycling</subject><subject>Tissue Fixation</subject><issn>0948-6143</issn><issn>1432-119X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNpdkctLxDAQxoMo7vo4epWA4K06eTbxJouPBcGLgreSJqmbpS-bVtj_3nZ3D-phGJj5zfDxfQhdELghAOltBOBEAQhgqThAc8IZTQjRH4doDpqrRI6TGTqJcQ1AhKb0GM0IcGCEkjlaLWscQz_gtik3le9M9NiuTKhx543tQ1NjUztsN7YM9Sduu1B5h8P-yFRtGYpgzQTe4VC1XfPtK1_3cXtmnGn77TKeoaPClNGf7_spen98eFs8Jy-vT8vF_UtimWJ9QkHCWEIJWRTEp0RxzqSWWlBmcpoTmjonbapyCoLl1uWOKypUDoxz6TQ7Rde7v6OUr8HHPqtCtL4sTe2bIWZSK0U1TODVP3DdDF09assoh1SMMshEJTvKdk2MnS-yyQLTbTIC2RRA9ieAkb_cfx3y0alf9M5x9gMBtn-c</recordid><startdate>19990501</startdate><enddate>19990501</enddate><creator>Paskins, L</creator><creator>Brownie, J</creator><creator>Bull, J</creator><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QP</scope><scope>7RV</scope><scope>7TK</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB0</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>NAPCQ</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope></search><sort><creationdate>19990501</creationdate><title>In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations</title><author>Paskins, L ; 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| ispartof | Histochemistry and cell biology, 1999-05, Vol.111 (5), p.411-416 |
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| subjects | Adaptation Azure Stains Bcl-2 protein Cell Line Chromosome 21 Chromosomes, Human, Pair 21 Color Enumeration Ethanol Fixatives Gene Dosage Humans Immunocytochemistry Labeling Microwaves Nucleotide sequence Polymerase chain reaction Primed In Situ Labeling - methods Staining and Labeling - methods Thermal cycling Tissue Fixation |
| title | In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations |
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