Basics of quantitative polymerase chain reaction: 2. Electrophoresis and quantitation of polymerase chain reaction products. 1996

C Sundfors, and Y Collan
Department of Pathology, University of Turku, Finland. carola.sund-fors@utu.fi

The performance of agarose and polyacrylamide (PAGE) gels in quantitating polymerase chain reaction (PCR) amplified c-erbB2 and p53 gene sequences (213 and 133 base pairs, respectively) was studied by applying image analysis on photographed ethidium bromide stained gels. The 5 mm thick agarose gels were more insensitive than the 1 mm thin PAGE gels and already started to show saturation effects within a narrow concentration range. The explanation is the greater dilution of band-associated products within the volume of the gel and the inefficient penetration of exciting UV light through the gel. Such effects produced inconsistency and dramatic variations in the band ratio estimates. In the system used by us, agarose electrophoresis and also electrophoresis using 5 mm thick PAGE gels have only a limited value in quantitation of PCR products with the band density ratios, but both can be used well in qualitative work. The far thinner (1 mm) PAGE gels, which did not markedly absorb UV light, performed better in the light of band density ratio estimates. The linear or approximately linear range of concentrations was wider. The coefficient of variation of band ratio, when estimated from the same gels, after loading them with aliquots of identical DNA from several PCR amplified tubes, was in the range of 4%. The absolute values of band densities had a more remarkable variation (than 4%) in both agarose and PAGE gels. Our recommendation is that quantitation of PCR products be done with band density ratio estimates, and in thin PAGE gels.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D004587 Electrophoresis, Agar Gel Electrophoresis in which agar or agarose gel is used as the diffusion medium. Electrophoresis, Agarose Gel,Agar Gel Electrophoresis,Agarose Gel Electrophoresis,Gel Electrophoresis, Agar,Gel Electrophoresis, Agarose
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D015203 Reproducibility of Results The statistical reproducibility of measurements (often in a clinical context), including the testing of instrumentation or techniques to obtain reproducible results. The concept includes reproducibility of physiological measurements, which may be used to develop rules to assess probability or prognosis, or response to a stimulus; reproducibility of occurrence of a condition; and reproducibility of experimental results. Reliability and Validity,Reliability of Result,Reproducibility Of Result,Reproducibility of Finding,Validity of Result,Validity of Results,Face Validity,Reliability (Epidemiology),Reliability of Results,Reproducibility of Findings,Test-Retest Reliability,Validity (Epidemiology),Finding Reproducibilities,Finding Reproducibility,Of Result, Reproducibility,Of Results, Reproducibility,Reliabilities, Test-Retest,Reliability, Test-Retest,Result Reliabilities,Result Reliability,Result Validities,Result Validity,Result, Reproducibility Of,Results, Reproducibility Of,Test Retest Reliability,Validity and Reliability,Validity, Face
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D016158 Genes, p53 Tumor suppressor genes located on the short arm of human chromosome 17 and coding for the phosphoprotein p53. Genes, TP53,TP53 Genes,p53 Genes,Gene, TP53,Gene, p53,TP53 Gene,p53 Gene
D018815 Genes, erbB Retrovirus-associated DNA sequences (erbB) originally isolated from, or related to, the avian erythroblastosis virus (AEV). These genes code for the epidermal growth factor receptor (EGFR) family of receptors which is important in the control of normal cell proliferation and in the pathogenesis of human cancer. The genes include erbB-1 (GENES, ERBB-1), erbB-2 (GENES, ERBB-2), and erbB-3, all of which show abnormalities of expression in various human neoplasms. c-erbB-3 Genes,erbB Genes,erbB-3 Genes,Genes, c-erbB-3,c erbB 3 Genes,c-erbB-3 Gene,erbB 3 Genes,erbB Gene,erbB-3 Gene

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