A Study of PCR Inhibition Mechanisms Using Real Time PCR
: In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting...
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| Veröffentlicht in: | Journal of forensic sciences 2010-01, Vol.55 (1), p.25-33 |
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| container_title | Journal of forensic sciences |
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| creator | Opel, Kerry L. Chung, Denise McCord, Bruce R. |
| description | : In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co‐extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance. |
| doi_str_mv | 10.1111/j.1556-4029.2009.01245.x |
| format | Article |
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Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co‐extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.</description><subject>Calcium</subject><subject>Chelating Agents</subject><subject>Collagen</subject><subject>Coloring Agents</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA Primers</subject><subject>DNA typing</subject><subject>forensic science</subject><subject>Forensic sciences</subject><subject>hematin</subject><subject>Hemin</subject><subject>Humans</subject><subject>humic acid</subject><subject>Humic Substances</subject><subject>HUMTH01</subject><subject>Indigo Carmine</subject><subject>Indoles</subject><subject>inhibition</subject><subject>Magnesium</subject><subject>melanin</subject><subject>Melanins</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Real time</subject><subject>real time polymerase chain reaction</subject><subject>Studies</subject><subject>Tannins</subject><subject>Taq Polymerase</subject><subject>Temperature</subject><issn>0022-1198</issn><issn>1556-4029</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkMtu1DAUQC0Eaoe2v4AsNqwSru3YsRcsyoiZFg0t6gOWVmLfUA95tPFEzPw9CVNmwQpvbMnnXF0dQiiDlI3n_TplUqokA25SDmBSYDyT6fYFmR0-XpIZAOcJY0Yfk9cxrgFAMcWOyPHoMMkUnxF9Tm83g9_RrqJf5zf0sn0IZdiErqVf0D0UbYhNpPcxtD_oDRY1vQsNTuQpeVUVdcSz5_uE3C8-3c0vktX18nJ-vkpcZoxMuMlKVQrM0fsSpXeZd7LU0qNHYKpC500B2rnKl4aXjjnM86qqDHgtNGhxQt7t5z723dOAcWObEB3WddFiN0SbC2HASAEj-fYfct0NfTsuZzkzygjI1AjpPeT6LsYeK_vYh6bod5aBndratZ0S2imhndraP23tdlTfPM8fygb9QfwbcwQ-7IFfocbdfw-2nxfX02v0k70f4ga3B7_of1qVi1za71dLuxTfLlYf9cJeid-KWpUp</recordid><startdate>201001</startdate><enddate>201001</enddate><creator>Opel, Kerry L.</creator><creator>Chung, Denise</creator><creator>McCord, Bruce R.</creator><general>Blackwell Publishing Ltd</general><general>Wiley Subscription Services, Inc</general><scope>BSCLL</scope><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>K7.</scope><scope>7X8</scope></search><sort><creationdate>201001</creationdate><title>A Study of PCR Inhibition Mechanisms Using Real Time PCR</title><author>Opel, Kerry L. ; Chung, Denise ; McCord, Bruce R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4995-294b6b3e7eddbe5dc4dc5b85dede016fecd9a08ccfdb92bc1ce77fff90d838083</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Calcium</topic><topic>Chelating Agents</topic><topic>Collagen</topic><topic>Coloring Agents</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA Primers</topic><topic>DNA typing</topic><topic>forensic science</topic><topic>Forensic sciences</topic><topic>hematin</topic><topic>Hemin</topic><topic>Humans</topic><topic>humic acid</topic><topic>Humic Substances</topic><topic>HUMTH01</topic><topic>Indigo Carmine</topic><topic>Indoles</topic><topic>inhibition</topic><topic>Magnesium</topic><topic>melanin</topic><topic>Melanins</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Real time</topic><topic>real time polymerase chain reaction</topic><topic>Studies</topic><topic>Tannins</topic><topic>Taq Polymerase</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Opel, Kerry L.</creatorcontrib><creatorcontrib>Chung, Denise</creatorcontrib><creatorcontrib>McCord, Bruce R.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Criminal Justice (Alumni)</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of forensic sciences</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Opel, Kerry L.</au><au>Chung, Denise</au><au>McCord, Bruce R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A Study of PCR Inhibition Mechanisms Using Real Time PCR</atitle><jtitle>Journal of forensic sciences</jtitle><addtitle>J Forensic Sci</addtitle><date>2010-01</date><risdate>2010</risdate><volume>55</volume><issue>1</issue><spage>25</spage><epage>33</epage><pages>25-33</pages><issn>0022-1198</issn><eissn>1556-4029</eissn><coden>JFSCAS</coden><abstract>: In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. 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| identifier | ISSN: 0022-1198 |
| ispartof | Journal of forensic sciences, 2010-01, Vol.55 (1), p.25-33 |
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| language | eng |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete |
| subjects | Calcium Chelating Agents Collagen Coloring Agents Deoxyribonucleic acid DNA DNA Primers DNA typing forensic science Forensic sciences hematin Hemin Humans humic acid Humic Substances HUMTH01 Indigo Carmine Indoles inhibition Magnesium melanin Melanins Polymerase Chain Reaction - methods Real time real time polymerase chain reaction Studies Tannins Taq Polymerase Temperature |
| title | A Study of PCR Inhibition Mechanisms Using Real Time PCR |
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