Method for quantitative measurement of gene expression using multiplex competitive reverse transcriptase-polymerase chain reaction

A technology for quantitative determination and gene expression, applied in enzyme production/bioreactors, specific-purpose bioreactors/fermentors, separation methods, etc., can solve the problems of sample addition error and inconvenience between tubes of amplification conditions

Inactive Publication Date: 2000-08-30
MEDICAL COLLEGE OF OHIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when two genes are amplified in separate tubes, tube-to-tube variability in amplification conditions and loading errors are unavoidable
Non-competitive multiplex PCR has also been described in Noonan, supra, i

Method used

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  • Method for quantitative measurement of gene expression using multiplex competitive reverse transcriptase-polymerase chain reaction
  • Method for quantitative measurement of gene expression using multiplex competitive reverse transcriptase-polymerase chain reaction
  • Method for quantitative measurement of gene expression using multiplex competitive reverse transcriptase-polymerase chain reaction

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Experimental program
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Effect test

preparation example Construction

[0052] Primers and internal competing templates for mutations were prepared as follows: The ideal sequence was prepared with Oligo -TM Primer analysis software (National Biosciences, Hamel, Mn.) identified. Primers were prepared using an Applied Biosystems Model 391 PCR-Mate DNA Synthesizer. The primer sequences are described below.

[0053] Glutathione peroxidase (GSH-Px) (Chada et al., Genomics 6:268-271, 1990)

[0054] The "outer" primers used to amplify native and mutated templates yielded products 354 bp long. The "outer" primer is

[0055] SEQ.I.D.No.1) (Chada et al., Genomics 6:268-271, 1990)

[0056] Position 241 5'-GGGCCTGGTGGTGCTTCGGCT-3' (encoding the sense strand) corresponds to bases 241-261 of the cloned sequence, and

[0057] SEQ.I.D.No.2) (Chada et al., Genomics 6:268-271, 1990)

[0058] Position 574 5'-CAATGGTCTGGAAGCGGCGGC-3' (encodes antisense strand), which anneals to bases 574-594.

[0059] The "internal" primer used to synthesize the mutated intern...

Embodiment

[0120] Serial dilutions of BEP2D cDNA were co-amplified with constant amounts of internal standard competition templates (10 attomoles each) for each single-base mutation before assaying. The negative of the gel ( figure 2 shown) were analyzed by densitometry to quantify the individual bands.

[0121] NN

N M

N M

MM

[0122] Wherein N = the ratio of single-stranded natural products before heavy annealing, M = the ratio of single-stranded mutant products before heavy annealing, NN (or N 2 ) = fraction of double-stranded natural products after heavy annealing, 2NM = fraction of heterodimers formed after heavy annealing, and MM (or M 2 ) = proportion of double-stranded mutation products after heavy annealing.

[0123] Heterodimers were counted indirectly because they were not restricted and had the same electrophoretic mobility as undigested homodimers. Therefore, heterodimers were detected by densitometry along with undigested homodimers. To q...

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Abstract

A method and apparatus for quantitative measurement of gene expression through multiplex competitive reverse transcriptase polymerase chain reaction amplification are disclosed. The method and apparatus are especially useful for analysis of small specimens of cells and tissues.

Description

technical background [0001] This application is a continuation-in-part of 08 / 043,390, filed April 6, 1993, and a continuation-in-part of 08 / 118,434, filed January 28, 1994. [0002] This invention was made with support from the National Institutes of Health Research Grants ES02679 and ES01247, the Department of Research Resources Grant No. RR00044, the Institute of Health Contract No. 91-2, and the International Lead and Zinc Organization Contract No. CH611 , who have certain rights in this invention. The present invention generally relates to methods for the quantitative determination of cellular levels of RNA by reverse transcription and polymerase chain reaction (PCR) amplification. [0003] PCR techniques are generally described in US Patents 4,683,195, 4,683,202, and 4,965,188. PCR technology generally includes methods for amplifying any desired specific nucleic acid sequence contained in a certain nucleic acid sequence in a certain nucleic acid molecule. The PCR metho...

Claims

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Application Information

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IPC IPC(8): C12N15/09B01D57/02B01L3/00B01L7/00C12M1/00C12M1/34C12M1/36C12M1/38C12M1/40C12Q1/68G01N33/50G01N33/53G01N35/10G01N37/00
CPCB01D57/02C12Q1/686C12Q1/6851C12Q2531/113C12Q2545/107C12Q2525/204C12Q2537/143C12Q2521/107
Inventor J·C·威利E·L·克劳福德J·P·德穆斯C·M·杰克逊D·A·韦弗
Owner MEDICAL COLLEGE OF OHIO
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