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Multi-local genomic analysis by method of improved cycle sequencing

A sequencing primer and nucleic acid sequencing technology, applied in the field of nucleic acid analysis, can solve problems such as diseases and cell collapse

Inactive Publication Date: 2002-01-02
BIO ID DIAGNOSTIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

cp strains cause cellular disruption and disease

Method used

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  • Multi-local genomic analysis by method of improved cycle sequencing
  • Multi-local genomic analysis by method of improved cycle sequencing
  • Multi-local genomic analysis by method of improved cycle sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Disclosed in this example are aspects of the invention that can be used to detect bovine viral diarrhea virus (BVDV). The method of this example can be adapted to detect and identify nucleic acids from other organisms, such as other RNA viruses.

[0058] The sequence analysis of this example employs a two-dye system. The system utilizes two lasers to detect the labeled products of its sequencing reactions. One laser was excited at a wavelength of 700nm and the other at a wavelength of 800nm. The primers of the sequencing reaction are labeled with two different near-infrared fluorescent dyes (such as dyes purchased from LI-COR, Inc. of Lincoln, Nebraska, U.S.A.), and one dye can respond to the irradiation of 700nm laser, which is recorded as IRD700 , another dye can react to 800nm ​​laser, denoted as IRD800. Using this system, two different DNA fragments of the same molecular size produced by different sequencing reactions but each labeled with a different dye can be ...

Embodiment approach

[0087] cDNA can be prepared from isolated RNA according to methods known in the art. One implementation is as follows:

[0088] a. Mix the following reagents in a 1.5ml centrifuge tube:

[0089] Total RNA 1-2μl;

[0090] Downstream primer (10pM / ml) 2μl;

[0091] Water 8μl

[0092] b. Heat the mixture to 70°C for 10 minutes;

[0093] c. Add the following reagents to the heated mixture:

[0094] 4 μl of 5× first-strand synthesis buffer;

[0095] dNTP (10mM) 1 μl; 0.1M DTT 2 μl;

[0096] d. Heat the mixture to 42°C for 2 minutes;

[0097] e. Add 1 μl SUPERSCRIFT II (a reverse transcriptase available from LifeTechnologies, Inc., Gaithersburg, Maryland, U.S.A.) to the above mixture;

[0098] f. 42 ℃ heat preservation for 50 minutes;

[0099] g. Heat to 70°C and hold for 10 minutes to terminate the reaction.

[0100] 3. Amplification of Specific Segments

[0101] Amplification of cDNA may be performed by any suitable method known to those skilled in the art. In the imple...

Embodiment 2

[0123] This example provides the analysis of four segments of the BVDV genome using primers of different molecular weights and different fluorescent labels. This embodiment is to use the dual-dye sequencing system described in Example 1 to resolve the co-migration sequencing reaction products produced by primer sets 1 and 2, and to distinguish the co-migration sequencing reaction products produced by primer sets 3 and 4, while the primer set The extension products of 1 and 2 were distinguished from the extension products of primer sets 3 and 4 by molecular weight. The primers used in this embodiment are as follows:

[0124] Primer set 1

[0125] [IRD700 labeled] 5'GTA GGT AGA GTG AAA CCC GG

[0126] (Hybridizes with the first strand of the BVDV genome at position 6941-6961)

[0127] [Unlabeled] 5' CGG GAC CTG GAC TTC ATA GC

[0128] (can hybridize with the complementary strand of the BVDV genome at position 7255-7245)

[0129] Primer set 1 can amplify a segment of 314 bas...

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Abstract

The invention provides a method of sequencing a nucleic acid in a reaction mixture comprising first and second nucleic acid target sequences. The target sequences may be present on the same or different nucleic acid molecules. First and second labelled sequencing primers are provided that are hybridizable, respectively, to the first and second nucleic acid target sequences. The second sequencing primer being at least as long as the total length of the first sequencing primer plus the length of the first target sequence. Each of the sequencing primer extension products beginning with the second sequencing primer will be longer than the primer extension products in the pool beginning with the first sequencing primer. Once the sequencing reactions have been completed, the lengths of the primer extension products in the pool may be detected by known methods, such as by gel electrophoresis.

Description

field of invention [0001] The present invention relates to methods for nucleic acid analysis, including methods that can be used for sequence analysis and diagnostic identification of cell types, such as pathological tissue identification. Background of the invention [0002] Various methods of nucleic acid sequencing are known. Most of the DNA sequencing methods used today are derived from Sanger's dideoxy chain termination method (Sanger, F., S. Nicklen and A.R. Coulson (1977) "Sequencing DNA with inhibitors capable of chain termination" PNAS USA74: 5463-5467). In these methods, under the catalysis of polymerase, the replication is initiated by a labeled primer complementary to a part of the strand to be sequenced. These methods typically require four sets of polymerase reactions, each using one of four dideoxynucleotides (ddNTPs) mixed with common deoxynucleotides. ddNTPs are unable to form phosphodiester bonds with subsequent nucleotides, so strand polymerization in e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/6869
CPCC12Q1/6869C12Q2565/125C12Q2535/113C12Q2535/101C12Q2525/204
Inventor T·维纳雅加莫尔蒂
Owner BIO ID DIAGNOSTIC
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