Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1994-06-08
1998-06-16
Zitomer, Stephanie W.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 435 911, 536 2433, 536 2532, 935 77, 935 78, C07H 2104, C12P 1934, C12Q 168
Patent
active
057668892
ABSTRACT:
Apparatus and method for determining an unknown starting molar concentration of target nucleic acid molecules at the beginning of a polymerase chain reaction in a sample reaction mixture containing suitable buffers, two complementary kinds of oligonucleotide primers, a molar excess of four kinds of nucleoside triphosphates, a DNA polymerase, and the unknown starting molar concentration of target nucleic acid molecules, wherein the two kinds of primers are provided in a known concentration (C.sub.po), the method including the steps of adding a reporter molecule which does not significantly interfere with the reaction and does not emit a strong signal in the absence of double-stranded DNA, but makes the dsDNA created by the reaction detectable and measurable at least once each cycle, thermal cycling one or more standard reaction mixtures which are substantially the same in every respect except they have known starting molar concentration of target mucleic acid molecules to obtain one or more growth curves with known starting molar concentrations of target nucleic acid molecules, exciting the reaction mixture during at least the extension portion of each cycle, detecting and measuring the intensity of the signal during at least the extension portion of each of the cycles, converting said intensity to molar concentration values of dsDNA and storing the molar concentration values for each of the extension portions of each of the cycles, generating a measured curve of molar concentration of dsDNA versus cycle number from the stored concentration values, using successive approximations, determining values of e.sub.v, e.sub.s, a, C.sub.z (n) and A.sub.z (n) which provide a best fit of the measured curve to the one or more known growth curves according to the following relation:
C.sub.n+1 =C.sub.n e.sub.v plus the minimum of ##EQU1##
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Aker David
Grimes Edwin T.
The Perkin-Elmer Corporation
Tran Paul B.
Zitomer Stephanie W.
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