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Methods and materials for detecting mutations in quasispecies having length polymorphismsMethods and materials for detecting mutations in quasispecies having length polymorphisms description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080233566, Methods and materials for detecting mutations in quasispecies having length polymorphisms. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/603,195, filed Aug. 20, 2004, and U.S. Provisional Application No. 60/603,337, filed Aug. 20, 2004. BACKGROUND OF THE INVENTIONThe present invention generally relates to methods and materials for detecting the presence or absence of a mutation of interest in a pathogen. The present invention also relates to particular methods and primers for determining the presence or absence of a mutation among multiple human immunodeficiency virus (HIV-1) quasispecies present in a sample from a single patient. The nucleic acid sequence of pathogens are often subject to a high mutation rate, giving rise to a variety of polymorphic variants. For example, human immunodeficiency virus, a member of the Lentivirus group of retroviruses, and the primary causative agent of Acquired Immune Deficiency Syndrome (AIDS), or AIDS-related complex (ARC), typically undergoes frequent mutation. The HIV-1 RNA genome comprises various genes that encode proteins necessary for the replication of HIV-1. Like all other retroviruses, it has an RNA genome which is replicated by means of the viral reverse transcriptase (RT) enzyme, which copies the single-stranded viral RNA genome into a double-stranded DNA/RNA hybrid, resulting in integration of the DNA provirus into the host cell genome. The RT enzyme lacks a 3′exonuclease activity which normally helps the “proof-reading” function of a polymerase enzyme to repair errors. Consequently, the RT enzyme makes at least one error during every transcription of 10,000 bases copied, resulting in errors that are responsible for the high mutation rate of HIV-1. The HIV-1 RNA genome also includes a gene that encodes the envelope glycoprotein (env), which consists of two principle subunits—the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. The gp41 subunit encodes transmembrane proteins that facilitate fusion of the HIV-1 virus to the outer cell membrane of CD4 cells. Because the HIV-1 env protein plays a critical role in the initial infection of CD4 cells, it has been a primary target in the search for drugs that can inhibit the interaction of proteins responsible for fusion of HIV-1 to cells, thereby inhibiting HIV infection. One particular target in the gp41 subunit of the HIV-1 env protein is the heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains, which have been shown to play a key role in facilitating the conformational changes required for fusion of viral and cellular membranes. Because many anti-retroviral drugs target the HIV-1 env protein in order to inhibit entry of HIV-1 into the cell, many mutations responsible for HIV-1 drug resistance arise in this region. Various drugs that are presently available to treat HIV fall into three different classes—nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs). Presently available anti-retroviral compounds used to treat AIDS suffer from certain disadvantages, including transient CD4 cell count effects, incomplete inhibition of viral replication, toxicity at prescribing doses, and emergence of resistant forms of the virus. Even with the advent of combination therapies, many patients remain unable to achieve or maintain complete viral suppression even with anti-retroviral compounds. As a result of incomplete viral suppression, coupled with the very high mutagenicity rate of HIV virus (due to the error-prone nature of the viral RT enzyme) and the genetic variability of the virus, many HIV variants with decreased drug susceptibility have arisen. For example, the use of Enfuvirtide (Enf, previously referred to as T-20), the first of a new class of anti-HIV drugs that inhibit fusion of HIV with a host cell, has resulted in the emergence of resistance mutations in the first heptad repeat domain of gp41 (HR1) that have been linked to T-20 treatment failure. By identifying mutations associated with HIV-1 drug resistance to specific anti-retroviral drugs before therapeutic intervention, the particular course of therapeutic intervention can be optimized by selecting and administering drugs to which the virus is most susceptible. Mutations can be detected by various techniques, the most direct and reliable of which is sequencing of the viral DNA (genotyping). In the case of clinical genotyping, where critical and even life-saving decisions relating to therapeutic intervention are made based on the genotyping results, confirmation of sequencing results by comparison with the sequence of the complementary strand of DNA is even more critical. The effectiveness of genotyping, and the ability to obtain bidirectional confirmation of sequence results is, however, compromised when multiple species of a pathogenic vector are present in a single patient sample. Because a patient sample containing mixed species of a pathogenic vector will contain multiple variants of the DNA sequence, sequencing will show multiple bases at a particular location and, in the case of insertion or deletion mutations, will show multiple bases at each location over an entire region of the DNA as a result of a shift in the reading frame, thus confounding the results and precluding complementary strand confirmation of the sequence and identification of clinically relevant mutations within that sequence. Pathogenic vectors that are present in the form of multiple species within a patient sample have therefore become increasingly refractory to clinical genotyping efforts, and have become a significant challenge to creating diagnostic assays to detect clinically relevant mutations, such as mutations that cause viral resistance to particular therapeutic drugs. Consequently, there is a need to develop more accurate and reliable genotyping methods that are amenable to complementary strand confirmation in clinical settings, and that are capable of detecting and identifying clinically relevant mutations in a pathogenic vector present in the form of multiple quasispecies within a patient sample. SUMMARY OF THE INVENTIONThe present invention provides improved methods and materials for clinical genotyping of a pathogen, such as HIV, present in a patient sample containing multiple quasispecies of the pathogen. In a particular aspect, the present invention relates to methods and materials for detecting the presence or absence of a mutation of interest in a pathogen present in a sample containing multiple quasispecies of the pathogen having mixed length polymorphisms, wherein the mutation of interest is located adjacent to the length polymorphism In a particular aspect, the present invention is directed to methods and materials for detecting the presence or absence of a mutation of interest in a patient sample containing mixed quasispecies of a pathogen, wherein the mutation of interest is located adjacent to a predetermined length polymorphism, such as an insertion mutation or a deletion mutation, which results in quasispecies of different nucleic acid sequence lengths. In a particular aspect, the present invention provides methods and primers for improved accuracy in genotyping an HIV-1 virus having length polymorphisms, which may be present in a patient sample containing mixed quasispecies. The improved methods and materials of the present invention may improve therapeutic intervention and treatment of infectious diseases, including, for example, AIDS. The methods and primers of the present invention were developed as a result of the initial discovery that amplification and sequencing of the entire HIV-1 env gene fails to provide reliable complementary strand confirmatory data necessary to identify and confirm the existence of important drug resistance associated mutations, identification of which is essential to therapeutic intervention. The gp41 region of HIV-1 env gene contains a first heptad repeat domain (HR1) and a second heptad repeat domain (HR2). The region surrounding the first heptad repeat domain (HR1) is subject to frequent insertion or deletion mutations, resulting in mixed HIV-1 populations having drug resistance mutations within the HR1 domain, but also containing multiple quasispecies having length polymorphisms. The presence of mixed length polymorphisms among quasispecies in a single patient sample confounds efforts to obtain confirmatory sequence of the complementary strand with primers covering a larger region, because the mixed length polymorphism mutation that occurs between one of the outer primers and the mutations of interest results in complementary primer extension products having sequences of different lengths in the region between the primers, which in turn results in overlayed and out of frame sequence signals being generated. The method of the present invention improves reliability of genotyping by sequencing the region of the mutation of interest, exclusive of the region containing the length polymorphism. The primer set may be used alone or in conjunction with a secondary primer set that includes the regions of variability to confirm genotypes for samples that cannot be reliably characterized using other sequencing primers. While the methods of the present invention were developed initially with respect to the HIV-1 gene, such methods are nevertheless applicable to any pathogen having length polymorphisms among multiple quasispecies. Accordingly, the present invention is directed to a method for detecting the presence or absence of a mutation of interest in the nucleic acid of a pathogen, such as HIV-1, wherein the mutation of interest is located adjacent to a length polymorphism defining multiple quasispecies of the pathogen, comprising:
a) obtaining from the patient sample a double-stranded DNA template encompassing the mutation of interest;
b) sequencing a first strand of a region of the DNA template containing the mutation of interest, exclusive of the length polymorphism;
c) sequencing a second strand of a region of the DNA template containing the mutation of interest, exclusive of the length polymorphism; and
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