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Method for determining the presence of extension products

A product, micro-sequencing technology, applied in biochemical equipment and methods, microbial determination/inspection, fermentation, etc.

Inactive Publication Date: 2006-03-08
BIOHIT OY FI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is not surprising, then, that the measurement of the time-dependent decay of fluorescence anisotropy can provide even more information on the rotational and diffusional motion of macromolecules

Method used

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  • Method for determining the presence of extension products
  • Method for determining the presence of extension products
  • Method for determining the presence of extension products

Examples

Experimental program
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Embodiment 1

[0051] SNP genotypes were determined by cycle micro-sequencing (cMS) technology modified from the original solid-phase micro-sequencing (US Patent 6,013,431). figure 2 The procedure of Example 1 is illustrated. Template DNA was extracted from frozen EDTA blood samples by conventional methods. The primers used for PCR amplification were sequences flanking the polymorphic SNP site, which in this case was the Factor V Leiden mutation (Arg506Gln, corresponding to a nucleotide change from G to A in the coding strand of genomic DNA) , which confers protein C resistance and increases the patient's risk of trombosis. The primer sequences were 5'-TCT CTT GAA GGA AATGCC CCA-3' and 5'-GGG CTA ATA GGA CTA CTT CTA-3'. Add 5 μL of template DNA (5 ng / μL) to 40 μL of the PCR master mix previously dispensed in a 96-well PCR plate. PCR amplification was performed in DNA polymerase reaction buffer and 25 μM of PCR primers, 200 μM of dNTPs and 0.5 units of thermostable DNA polymerase were add...

Embodiment 2

[0055] DNA used as template was extracted from frozen EDTA blood samples by conventional methods. The primers used for PCR amplification were sequences flanking the polymorphic SNP site, which in this case was the Factor V Leiden mutation (Arg506Gln, corresponding to a nucleotide change from G to A in the coding strand of genomic DNA) , which confers protein C resistance and increases the patient's risk of trombosis. The primer sequences were 5'-TCT CTT GAA GGA AAT GCC CCA-3' and 5'-GGG CTA ATA GGA CTACTT CTA-3'. Add 5 μL of template DNA (5 ng / μL) to 40 μL of the PCR master mix previously dispensed in a 96-well PCR plate. PCR amplification was performed in DNA polymerase reaction buffer, and 25 μM of PCR primers, 200 μM of dNTPs and 0.5 units of thermostable DNA polymerase were added. The amplification reaction was carried out in an MJ Research Tetrad cycler with a dynamic drop PCR program at 64°C => 58°C: initially at 96°C for 5 minutes, then at 96°C for 1 minute, and withi...

Embodiment 3

[0059] We tested a variety of fluorescent dyes as acceptors to receive FRET energy from ssDNA-binding fluorescent reagent OliGreen (Molecular Probes, Eugene, OR). We took 5 μL of single-stranded DNA-specific fluorescent reagent OliGreen diluted 1:50 with TE buffer (10mM Tris-HCl, pH 7.0, 1mM EDTA), and added it to the sample containing 5pmol of primers labeled with one of the following dyes: Tetramethyl Rhodamine-based (emit around 552nm), Texas Red TM (emit around 620nm, Molecular Probe, Eugene OR), Cy3 (emit around 570nm, Amersham Lifescience, Inc.) or Cy5 TM (emission around 667nm, Amersham Life Science, Inc.). All of these dyes emit at their expected emission wavelengths when attached to oligonucleotides, and are excited at 485 nm in the presence of OliGreen bound to the primer. Thus, using ssDNA-binding fluorescent reagents and FRET, the presence of multiple oligonucleotides carrying fluorophores with different emission peaks can be detected.

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Abstract

The present invention relates to a method and reagents for determining extension products in reactions involving nucleic acid chain extension, e.g., in cyclic minisequencing, and to the use of this method when identifying specific point mutations and genetic variations. Three standard unmodified primers are needed and a non-specific fluorescent reagent (e.g. a ssDNA-binding dye) can be used as one of the fluorophores in the FRET assay used. The invention is also related to vertical light beam fluorometry.

Description

field of invention [0001] The present invention relates to a method and reagents for determining extension products produced in reactions involving nucleic acid chain extension, such as cyclic minisequencing, and to applications of the method in detecting specific point mutations and genetic variations. Background of the invention [0002] Point mutations, changes to a single nucleotide in a gene, are a major cause of genetic disease in humans. For diagnostic purposes, there are many techniques suitable for detecting known point mutations. These methods can be classified as those relying on differential hybridization kinetics between fully and partially complementary DNA strands, or relying on enzyme-assisted point mutation detection (Landegren, et al., 1998). [0003] The classic ASO (allele-specific oligonucleotide hybridization) (Wallace, et al., 1979) using radioactively labeled oligonucleotide probes has become a kinetic homogeneity analysis system after years of devel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34C12Q1/6858C12Q1/6869G01N
CPCC12Q1/6869C12Q1/6858C12Q2565/101C12Q2533/101
Inventor 阿托·奥帕纳奥斯莫·斯沃瓦尼米
Owner BIOHIT OY FI
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