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Genotyping method

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Genotyping method


The present invention relates to a genotyping method, and more particularly to an ID sequence, which is assigned to each genotype, and a multiplex genotyping method which uses the ID sequence. When pyrosequencing is performed using the ID sequence, a unique and simple pyrogram can be obtained for each genotype. Thus, the use of the ID sequence makes it possible to genotype viral genes, disease genes, bacterial genes and identification genes in a simple and efficient manner. In addition, a genotyping primer of the invention can be used in various genotyping methods which are performed using dispensation orders and sequencing methods.
Related Terms: Genotype

Browse recent Genomictree, Inc. patents - Daejeon, KR
Inventors: Sung Whan An, Myung Sok Oh
USPTO Applicaton #: #20120276524 - Class: 435 5 (USPTO) - 11/01/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Virus Or Bacteriophage

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The Patent Description & Claims data below is from USPTO Patent Application 20120276524, Genotyping method.

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TECHNICAL FIELD

The present invention relates to a genotyping method, and more particularly to an ID sequence, which is assigned to each genotype, and a multiplex genotyping method which uses the ID sequence.

BACKGROUND ART

Methods which have been developed for detecting infectious organisms include traditional methods of identifying the physical and chemical characteristics of pathogens by cultivation, and methods of detecting the specific genetic characteristics of pathogens. The methods for detecting genetic characteristics include restriction fragment length polymorphism (RFLP) analysis, amplified fragment length polymorphism (AFLP) analysis, pulsed-field gel electrophoresis, arbitrarily-primed polymerase chain reaction (AP-PCR), repetitive sequence-based PCR, ribotyping, and comparative nucleic acid sequencing. These methods are generally too slow, expensive, irreproducible, and technically demanding to be used in most diagnostic settings. All of the above-mentioned methods generally require that a cumbersome gel electrophoretic step be used, that the pathogen be grown in culture, that its genomic DNA be purified, and that the sample not contain more than one type of organism. These limitations also apply to recently developed detection methods which employ high density microarrays (Salazar et al., Nucleic Acids Res. 24:5056-5057, 1996; Troesch et al., J. Clin. Microbiol. 37:49-55, 1999; Lashkari et al., Proc. Natil. Acad. Sci. U.S.A. 94: 13057-13062, 1997). Meanwhile, pyrosequencing is a method of DNA sequencing based on the “sequencing by DNA synthesis” principle, which relies on the detection of pyrophosphate release on nucleotide incorporation, unlike the traditional Sanger sequencing method. In the pyrosequencing method, four deoxynucleotide triphosphates (dNTPs) are sequentially added one by one during polymerization. PPi attached to the dNTPs being polymerized emit light by enzymatic reactions, and the emitted light shows a signal peak according to the reaction order of each of the sequentially added dNTPs, in which the peak shows a pattern which is high or low in proportion to the number of the reacted dNTPs, such that the nucleotide sequence of the pathogen can be determined. In recent years, methods of detecting pathogenic bacteria or viruses in clinical samples based on pyrograms obtained by pyrosequencing of the PCR products of sequences specific to the pathogens have been used (Travasso, C M et al, J. Biosci., 33:73-80, 2008; Gharizadeh, B et al., Molecular and Cellular Probes, 20, 230-238, 2006; Hoffmann, C et al., Nucleic Acid Research, 1-8, 2007).

In the pyrosequencing technique, however, nucleotide sequencing is performed according to the dispensation order of dNTPs, and a nucleotide in a template, which is absent in the dispensation order, does not react, and thus does not form a peak. However, when identical nucleotides in the dispensation order continuously appear, the heights of the peaks are determined according to the intensities of light emitted. Accordingly, when various pathogens exist in the same sample, the peaks of the nucleotides of the various pathogens appear overlapped, thus making it difficult to identify the genotypes through the interpretation of pyrograms. Particularly, as the number of repetitive sequences increases, the peaks of the anterior sequences become relatively lower. Thus, in the case of infection with multiple pathogens, it is difficult to detect a peak according to the degree of infection with each pathogen.

Accordingly, the present inventors have made extensive efforts to enable the genotypes of interest to be identified by unique and simple pyrograms obtained when performing genotyping using pyrosequencing. As a result, the present inventors have found that, when an ID sequence, which has an ID mark, a signpost and an endmark while existing independently of the specific sequence to be typed, is linked with the specific sequence and is used to perform pyrosequencing, a unique and simple pyrogram can be obtained for each genotype, thereby completing the present invention.

DISCLOSURE OF INVENTION

It is an object of the present invention to provide an ID sequence which is useful to perform pyrosequencing so as to enable the genotypes of interest to be identified by unique and simple pyrograms.

Another object of the present invention is to provide a genotyping method which uses said ID sequence.

Still another object of the present invention is to provide a method of genotyping HPV using said ID sequence.

Yet another object of the present invention is to provide a method of detecting KRAS gene mutation using said ID sequence.

A further object of the present invention is to provide a method of detecting respiratory virus using said ID sequence.

To achieve the above objects, the present invention provides an ID sequence for genotyping which consists of A(ID−S)n−E, wherein ID is an ID mark which is a single nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark; E is an endmark which is a nucleotide different from that of the signpost; and n is a natural number ranging from 1 to 32.

The present invention also provides an ID sequence for genotyping which consists of ID−S, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G, and S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark.

The present invention also provides a genotyping primer comprising a gene-specific sequence for genotyping linked to said ID sequence.

The present invention also provides a genotyping method which comprises using said genotyping primer.

The present invention also provides a method for genotyping HPV, the method comprising the steps of: (a) designing an ID sequence for genotyping according to the genotype of each HPV virus, the ID sequence consisting of (ID−S)n−E, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark; E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) constructing a genotyping primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific to a virus genotype corresponding to the ID sequence; (c) amplifying an HPV virus-containing sample by PCR using the genotyping primer; and (d) subjecting the amplified PCR product to pyrosequencing to obtain a sequence for the ID sequence, and distinguishing the genotype of HPV according to the ID sequence.

The present invention also provides a method for detecting KRAS gene mutation, the method comprising the steps of: (a) designing an ID sequence for genotyping according to the gene mutation of each KRAS, the ID sequence consisting of (ID−S)n−E wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark; E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) constructing a detection primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific for a KRAS gene mutation corresponding to the ID sequence; (c) amplifying a KRAS gene-containing sample by PCR using the detection primer; and (d) subjecting the amplified PCR product to pyrosequencing to obtain a pyrogram for the ID sequence, and detecting the KRAS gene mutation according to the ID sequence.

The present invention also provides a method for detecting respiratory virus, the method comprising the steps of: (a) designing an ID sequence for genotyping according to the genotype of each of influenza A virus, influenza B virus, RSV B, rhinovirus, and coronavirus OC43, the ID sequence consisting of (ID−S)n−E wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost, and n is a natural number ranging from 1 to 32; (b) constructing a detection primer composed of a pyrosequencing primer sequence, the ID sequence, and a sequence specific to each respiratory virus gene corresponding to the ID sequence; (c) amplifying a sample, which contains a respiratory virus selected from the group consisting of influenza A virus, influenza B virus, RSV B, rhinovirus, and coronavirus OC43, by PCR using the detection primer; and (d) subjecting the amplified PCR product to pyrosequencing to obtain a pyrogram for the ID sequence, and detecting the respiratory virus according to the ID sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a pyrosequencing process, which is performed according a dispensation order, and the resulting pyrogram.

FIG. 2 shows the change in pyrogram peaks according to analytical sequences.

FIG. 3 shows the change in pyrogram peaks according to analytical sequences.

FIG. 4 shows the change in pyrogram peaks, which results from insertion of a signpost.

FIG. 5 shows pyrograms obtained for a mixture of two different analytical sequences.

FIG. 6 shows pyrograms obtained in the presence or absence of a signpost in a dispensation order.

FIG. 7 shows the changes in pyrogram patterns according to the changes in a sequence posterior to a signpost.

FIG. 8 shows pyrograms obtained in the absence of an endmark.

FIG. 9 shows pyrograms obtained in the absence of an endmark.

FIG. 10 shows the change in the dispensation order according to the change in the order of a signpost.

FIG. 11 shows a method of designing an ID sequence according to a dispensation order.

FIG. 12 shows a method of designing a dispensation order.

FIG. 13 shows pyrograms obtained by ID sequences according to dispensation orders.

FIG. 14 shows a method of designing an ID sequence after determining a dispensation order.

FIG. 15 shows pyrograms obtained by ID sequences according to dispensation orders.

FIG. 16 shows a method of genotyping HPV using an ID sequence of the present invention.

FIG. 17 shows a general system for detecting KRAS mutations.

FIG. 18 shows a method of detecting KRAS mutations using ID sequences of the present invention.

FIG. 19 shows the results obtained by genotyping 15 HPV types using ID sequences of the present invention.

FIG. 20 shows the results obtained by genotyping two or more types of HPV.

FIG. 21 shows the results of detecting KRAS mutations using ID sequences of the present invention.

FIG. 22 shows the results of detecting multiple KRAS mutations using ID sequences of the present invention.

FIG. 23 shows the results of detecting KRAS mutations in colorectal cancer tissue using ID sequences of the present invention.

FIG. 24 shows a method of detecting respiratory virus infection using an ID sequence of the present invention.

FIG. 25 shows the results of detecting single infections of 5 types of respiratory viruses using ID sequences of the present invention.

FIG. 26 shows the results of detecting multiple infections of 5 types of respiratory viruses using ID sequences of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Other features and embodiments of the present invention will be more apparent from the following detailed descriptions and the appended claims

In one aspect, the present invention is directed to an ID sequence for genotyping which consists of A(ID−S)n−E, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G; S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark, E is an endmark which is a nucleotide different from that of the signpost; and n is a natural number ranging from 1 to 32.

In another aspect, the present invention is directed to an ID sequence for genotyping which consists of ID−S, wherein ID is an ID mark which is a nucleotide selected from among A, T, C and G, and S is a signpost which is a nucleotide linked with the adjacent ID mark and different from that of the adjacent ID mark.

As used herein, the term “ID sequence” is not a specific sequence conserved in each gene and refers to an artificially constructed nucleotide sequence which can be specifically assigned to each genotype in the genotyping method of the present invention.

As used herein, the term “adjacent ID mark” means an ID mark located ahead of or behind the signpost.



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stats Patent Info
Application #
US 20120276524 A1
Publish Date
11/01/2012
Document #
File Date
10/31/2014
USPTO Class
Other USPTO Classes
International Class
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Genotype


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