Method for mutation detection in HIV-1 using pol sequencing

Details Method for mutation detection in HIV-1 using pol sequencing Method for mutation detection in HIV-1 using pol sequencing

2000-08-18

2004-10-05

Winkler, Ulrike (Department: 1648)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S456000, C514S04400A, C536S023100, C536S023720, C536S024300, C536S024330

Reexamination Certificate

active

06800463

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATIONS
This Application claims priority to European Application No. 00201433.0, filed Apr. 20, 2000, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to a method for detecting mutations within the HIV pol gene of HIV-1 isolates and in particular with the design of amplification primers and sequencing primers for use in the analysis of the coding domains for the protease and reverse transcriptase, respectively.
BACKGROUND OF THE INVENTION
The availability of rapid, high-throughput automated DNA sequencing technology has obvious applications in clinical research, including the detection of variations in virus populations and mutations responsible for drug resistance in virus genomes. However, analysis of clinical samples by manual sequencing or polymerase chain reaction-(PCR) based point mutation assays has revealed that complex mixtures of wild type and mutant HIV-1 genomes can occur during drug therapy. Therefore, to assess the likely susceptibility of a virus population to a particular drug therapy, it would be desirable to perform DNA sequence analysis that can simultaneously quantitate several resistance mutations in multiple genomes. A particular advantage of analyzing the sequence of more than one pol gene enzyme (Protease and Reverse transcriptase) is that the studied material reflects to a greater extent the viral genetic diversity in the particular patient being investigated.
SUMMARY OF THE INVENTION
The aim of the present invention is thus to provide a reliable sequence analysis method and kit for performing mutation analysis of the pol gene of HIV-1 virus isolates.
In one embodiment, the present invention relates to a method for mutation analysis of the HIV pol gene of a HIV-1 virion comprising the steps of:
a) isolation of a sample comprising HIV-1 RNA,
b) amplifying RNA using outer primers as represented in SEQ ID No: 1 (OUT3) and 2 (PRTO-5),
c) amplifying the product of (b) using a 5′ and 3′ primer chosen from the inner primers as represented in SEQ ID No: 3 (PCR2.5), 4 (PCR2.3), 5 (SK107) and 6 (SK108), and
d) sequencing this secondary obtained product.
The present invention also provides a method for mutation analysis of the HIV pol gene of HIV-1 isolates comprising the steps of:
a) isolation of a sample comprising HIV-1 DNA,
b) amplifying DNA using outer primers as represented in SEQ ID No: 1 (OUT3) and 2 (PRTO-5),
c) amplifying the product of (b) using a 5′ and 3′ primer chosen from the inner primers as represented in SEQ ID No: 3 (PCR2.5), 4 (PCR2.3), 5 (SK107) and 6 (SK108), and
d) sequencing this secondary obtained product.
The present invention also relates to a primer as described herein (see Table 1) and used to analyze the sequence of the HIV pol gene of HIV-1 isolates. In a further embodiment, the present invention relates to a diagnostic kit for the mutation analysis of the HIV pol gene of HIV-1 isolates comprising at least one of the primers as shown in Table 1.
Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.


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