Novel methods for HIV sequencing and genotyping

Abstract

Methods are provided for amplifying regions of the HIV pol gene amplifying double-stranded nucleic acid template derived from HIV tube RT-PCR with novel PCR primers to produce amplified target sequences. Methods are also provided for analyzing the nucleotide sequence of these amplified targets using novel sequencing primers and the data is analyzed. The determined nucleotide sequence can be compared to the sequence of known drug resistance mutations in the HIV pot gene to determine the viral genotype.

Description

BACKGROUND AND SUMMARY OF THE INVENTION [0001] The present invention relates to methods for sequencing the Human Immunodeficiency Virus (HIV) nucleic acids. More specifically, the present invention relates to methods for obtaining information on the genetic sequences of HIV nucleic acid from a patient. That information can be used to genotype a HIV quasi-species present in the patient. [0002] The detection of mutations conferring drug resistance in the HIV pol gene is significant in determining drug sensitivity of the virus. During the course of treatment of a disease, the infectious microorganism or virus, such as HIV, can become resistant due to a loss of sensitivity to the particular drug in use, which generally-results in spread of the disease and increased morbidity. At the genetic level, important changes can occur within the virus in response to drug therapy. Specific changes in a nucleic sequence or nucleic acid sequences of the virus that correlate with drug resistance are ...

AI Technical Summary

Benefits of technology

[0016] According to certain preferred embodiments, the inventors have achieved improved sensitivity and determined a greater number of HIV quasi-species than procedures that employ nested primers. Certain embodiments involve a stream-lined assay, including a single-tube two step amplification procedure, coupled with automated sequence analysis and correlation with known drug resistance mutations. Such embodiments provide rapid, reliable assays that have acceptable sensitivity and specificity.

[0025] Double stranded DNA template is then created from the prepared HIV RNA. According to certain embodiments, random hexamers are used as primers at the 3′ end in conjunction with a reverse transcriptase (RT) procedure. An advantage of using random hexamers is that sequence variability will not affect cDNA generation. Random hexamers may also stabilize RNA secondary structure, which is known to be quite significant in HIV pol RNA. Appropriate conditions are used that result in cDNA generation from purified HIV RNA. In certain preferred embodiments, the double stranded templates produced in the RT reaction are then amplified in a PCR amplification method.

[0035] According to certain embodiments of the sequencing procedure, the new dye-terminator chemistries (dRhodamine and Big-Dye) were employed in place of dye-labeled primers. The new dye chemistries allow for more even incorporation of nucleotides and a much improved signal to noise ratio over the rhodamine terminators. The new Big-Dye terminator (U.S. Pat. No. 5,800,996) was chosen over the dRhodamine terminators because of the increased signal strength and better signal to noise ratio. This allows for a faster throughput for sequencing with increased data quality.

Problems solved by technology

During the course of treatment of a disease, the infectious microorganism or virus, such as HIV, can become resistant due to a loss of sensitivity to the particular drug in use, which generally-results in spread of the disease and increased morbidity.

This is due to the lack of fidelity and proof-reading functions by the virus's RNA polymerase or reverse transcriptase for retroviruses.

The high degree of enzyme-induced genetic variability, in addition to the selective pressures of drug therapy, makes genotypic assessment of HIV very complex.

Thus, physicians typically can only diagnose drug resistance in a patient if the patient fails to respond to therapy.

Moreover, without genetic analysis, if a patient is failing therapy, it is difficult, if not impossible, to determine for which drugs the patient is still sensitive.

Because of a high rate of mutation in HIV, technologies using labeled oligonucleotides to represent “mutant” or “wild-type” forms at a particular codon of the gene sequence will be adversely affected.

For example, mutations which are not associated with drug resistance will frequently occur, and may affect the binding of either “wild-type” or “mutant” probes, giving an anomalous result.

Although a variety of home brew sequencing based assays have been used in individual research labs, the present inventors are not aware of comprehensive, commercially available systems for determining the de novo sequence of infectious organisms.

The general poor quality and lack of proper controls, seen with most home brew assays, have hindered generation of accurate data which are crucial for studying drug resistance.

While this procedure is effective at amplifying a known sequence present at low copy number, the use of multiple sets of primers, each of which must hybridize successively to a target sequence in the gene of interest, can result in a loss of the ability to amplify highly variable nucleic acid sequences.

This results in biased selection of HIV quasi-species wherein significant drug resistance mutations may remain entirely undetected until drug failure occurs in the patient.

Among such quasi-species, there are likely to be mutations due to drug resistance as well as random mutations due to polymerase error in the virus population.