Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-06-25
2003-01-07
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007100, C435S021000, C435S028000, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06503704
ABSTRACT:
This application relates to a method, reagent and kit for genotyping of human papillomavirus, and in particular to the sequencing of human papillomavirus for determination of viral type.
Cancer of the cervix is one of the most common malignancies in women around the world. Over 90% of both invasive cervical cancer lesions and precursor lesions are associated with the presence of human papillomavirus (HPV), and many epidemiological studies have established that HPV infection is the major risk factor for squamous intraepithelial lesions and cervical carcinoma. Recently, the involvement of HPV in the etiology of cervical cancer has been extended to prostate cancer. Epidemiological studies have shown that men with HPV infections in their 20's and 30's are five times more likely to develop prostate cancer in their 50's and 60's.
In view of the potential significance of HPV infection, it would clearly be of interest to be able to routinely test samples for the presence of HPV. However, of the more than 54 genetic types of HPV which have been described (an HPV isolate is designated as a new “type” when it has less than 90% nucleotide homology in the E6, E& and L1 genes with previously characterized HPV types), only about 20% have been shown to be oncogenic. Thus, it is not sufficient to detect HPV. Meaningful diagnosis also requires the determination of the genetic type of any infecting virus.
U.S. Pat. No. 5,447,839, which is incorporated herein by reference, discloses a method for detection and typing of HPV. In this method, HPV DNA sequences in a sample are amplified by polymerase chain reaction (PCR) amplification using consensus primers which amplify both oncogenic and non-oncogenic HPV types. Thus, the presence of HPV in the sample is indicated by the formation of amplification products. HPV is then typed using type-specific DNA probes which hybridize with the amplified region of DNA. The type-specific hybridization probes disclosed in this patent are capable of identifying and distinguishing among five known oncogenic types of HPV, namely HPV-6, HPV-11, HPV-16, HPV-18 and HPV-33.
U.S. Pat. Nos. 4,849,331, 4,849,332, 4,849,334 and 4,908,306 which are incorporated herein by reference relate to HPV-35, HPV-43, HPV-44, and HPV-56. According to these patents, these types may be identified by hybridization with type-specific probes, although no actual sequences for such probes are disclosed.
Identification of other HPV types is discussed in Schiffman, et al. (1993). “Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia”,
J. Nat'l Cancer Inst
. 85: 958-964; zur Hausen, H., (1994) “Molecular pathogenesis of cancer of the cervix and its causation by specific human papillomavirus types”,
Curr. Top. Microbiol. Immunol
. 186: 131-156; and de Villiers, E. (1994). “Human pathogenic papillomavirus types: an update”,
Curr. Top. Microbiol. Immunol.
186: 1-12.
What is apparent from consideration of the art discussed above is that determination of HPV type using hybridization probes requires a substantial arsenal of distinct probes types, and a battery of tests which makes HPV typing by this approach both time consuming and expensive. Furthermore, since the number of identified types of HPV is continuing to expand, there is a need to keep developing new tests and reagents and a risk that an existing hybridization probe is in fact unable to distinguish between a known genotype and a yet-to-be characterized genotype. Thus, it would be advantageous to perform the genotyping of HPV samples using reagents that are not type-specific. It is an object of the present invention to provide such a method.
SUMMARY OF THE INVENTION
This and other objects of the invention are achieved using a method for determining the sequence of human papillomavirus present in a sample comprising the steps of:
(a) amplifying a portion of the L1 open reading frame of human papillomavirus genome to form L1 amplicons containing plus and minus amplified strands using first and second amplification primers; and
(b) determining the positions of at least one species of nucleotide within at least one of the plus and minus amplified strands by extension of a sequencing primer which hybridizes with the plus or minus amplified strand in the presence of a chain-terminating nucleotide,
wherein the first amplification primer has the sequence given by Seq. ID. No. 1, and the sequencing primer has the sequence given by Seq. ID No. 3. The second amplification primer preferably has the sequence given by sequence ID No. 2.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for sequencing, and thus for determining the genotype of HPV that may be present in a sample. Suitable samples for use in the present invention include but are not limited to cervical swabs or scrapings, urethral swabs, vaginal/vulval swabs, urine and biopsied tissues samples.
In accordance with the present invention, a sample containing, or suspected of containing HPV is combined with a pair of amplification primers effective to amplify a portion of the L1 open reading frame of the HPV genome via polymerase chain reaction (PCR) amplification. The procedures for PCR amplification have become well known, and will not be repeated at length here. Basically, however the two primers are selected to flank a region of interest to be amplified, one primer binding to each of the strands of the DNA duplex such that template-dependent primer extension proceeds in the direction of the other primer binding site. Repeated cycles of annealing, extension and denaturation result in the production of many copies of both the plus and minus (sense and antisense) strands in the region flanked by the primers. The double stranded copies of the L1 region are referred to herein as L1 amplicons. Each such amplicon of course contains a plus and a minus strand.
Consensus amplification primer sequences for the L1 open reading frame of HPV have been previously described in U.S. Pat. No. 5,447,839 and in Ting et al., “Detection and Typing of Genital Human Papillomavirus”,
PCR Protocols: A Guide to Methods and Applications
, Academic Press, 1990, pp. 356-367. These primers, designated as MY11 and MY09, respectively, have the following sequences:
MY11, positive strand primer:
CGCMCAGGGWC ATAAYAATGG Seq. ID. No. 1
MY09, negative strand primer:
CGTCCMARRG GAWACTGATC Seq. ID No. 2.
A third primer, HMB01 (SEQ ID No. 4) is often used in combination with MY09 and MY11 to amplify HPV 51 which is not amplified efficiently with MY09 and MY11 alone. Hildesheim et al.,
J. Infect. Dis
. 169: 235-240 (1994). This amplification primer, or other additional primers which increase amplification efficiency for difficult types may be included in amplification mixtures when practicing the present invention. See Qu et al. (1997)
J. Clin. Microbiol
. 35: 1304-1310. In a preferred embodiment of the present invention, the MY11, MY09 and HMB01primers are used to amplify HPV that may be present in the sample to be tested. This results in the production of L1 amplicons.
The next step in the method of the invention is the determination of the nucleic acid sequence of at least the minus strand of the L1 amplicons. This is accomplished using a chain termination sequencing method and a sequencing primer having the sequence:
ARRGGAWACT GATCWARDTC Seq. ID No. 3.
Like PCR, chain termination nucleic acid sequencing is a well known procedure, although many variations have been developed. In the basic procedure for chain-termination sequencing, a polynucleotide to be sequenced is isolated, rendered single stranded if necessary, and placed into four vessels. In each vessel are the necessary components to replicate the DNA strand, i.e., a template-dependant DNA polymerase, a short primer molecule complementary to a known region of the DNA to be sequenced, and the standard deoxynucleotide triphosphates (dNTP's) commonly represented by A, C, G and T, in a buffer conducive to hybridization between the primer and the DNA to b
Chong Sylvia
Kierstead Timothy
Mahony James
Seadler Alan
Chakrabarti Arun Kr.
Jones W. Gary
Oppedahl & Larson LLP
Visible Genetics Inc.
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