U.S. patent application number 09/802110 was filed with the patent office on 2003-05-01 for method, compositions and kit for detection of microorganisms and bi-directional sequencing of nucleic acid polymers.
Invention is credited to Dunn, James M., Hui, May, Lacroix, Jean-Michel, Leushner, James.
Application Number | 20030082535 09/802110 |
Document ID | / |
Family ID | 27533344 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082535 |
Kind Code |
A1 |
Leushner, James ; et
al. |
May 1, 2003 |
Method, compositions and kit for detection of microorganisms and
bi-directional sequencing of nucleic acid polymers
Abstract
Evaluation of a sample for the presence and qualitative nature
of a microorganism can be performed in a single vessel by combining
a natural abundance DNA sample with a sequencing mixture containing
a primer pair, a thermally stable polymerase such as
ThermoSequenase.TM. which incorporates dideoxynucleotides into an
extending nucleic acid polymer at a rate which is no less than
about 0.4 times the rate of incorporation of deoxynucleotides,
nucleotide triphosphate feedstocks, and a chain terminating
nucleotide triphosphate. The mixture is processed through multiple
thermal cycles for annealing, extension and denaturation to produce
a product mixture which is analyzed by electrophoresis.
Inventors: |
Leushner, James; (North
York, CA) ; Hui, May; (Toronto, CA) ; Dunn,
James M.; (Scarborough, CA) ; Lacroix,
Jean-Michel; (Etobicoke, CA) |
Correspondence
Address: |
OPPEDAHL AND LARSON LLP
P O BOX 5068
DILLON
CO
80435-5068
US
|
Family ID: |
27533344 |
Appl. No.: |
09/802110 |
Filed: |
March 7, 2001 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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09802110 |
Mar 7, 2001 |
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09311260 |
May 13, 1999 |
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6214555 |
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09311260 |
May 13, 1999 |
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09009483 |
Jan 20, 1998 |
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6083699 |
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09311260 |
May 13, 1999 |
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08640672 |
May 1, 1996 |
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5789168 |
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09311260 |
May 13, 1999 |
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08684498 |
Jul 19, 1996 |
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5830657 |
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09311260 |
May 13, 1999 |
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08807138 |
Feb 27, 1997 |
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5888736 |
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09311260 |
May 13, 1999 |
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PCT/US97/07134 |
Apr 29, 1997 |
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Current U.S.
Class: |
435/6.15 ;
435/91.2 |
Current CPC
Class: |
C12Q 2565/102 20130101;
C12Q 2535/113 20130101; C12Q 1/6869 20130101; C12Q 1/6869 20130101;
B01L 7/52 20130101; C12Q 1/703 20130101; C12Q 1/689 20130101; G01N
2035/00237 20130101; C12Q 1/6841 20130101; B01L 7/525 20130101;
C12Q 1/6881 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
1. A composition comprising a mixture of four deoxynucleotide
triphosphates and at least one dideoxynucleotide triphosphate
corresponding to one of the four deoxynucleotide triphosphates,
wherein the dideoxynucleotide triphosphate is present in a mole
ratio to the corresponding deoxynucleotide triphosphate of from
1:50 to 1:500, said composition further comprising a thermally
stable polymerase enzyme which incorporates dideoxynucleotides into
an extending nucleic acid polymer at a rate which is no less than
0.4 times the rate of incorporation of deoxynucleotides.
2. The composition according to claim 1, wherein the mole ratio is
from 1:100 to 1:300.
3. A kit for detection of a target microorganism comprising, in
packaged combination, (a) a pair of primers which bind to the sense
and antisense strands, respectively, and flank a selected region
within the genome target microorganism; and (b) a mixture of four
deoxynucleotide triphosphates and at least dideoxynucleotide
triphosphate corresponding to one of the four deoxynucleotide
triphosphates, wherein the dideoxynucleotide triphosphate is
present in a mole ratio to the corresponding deoxynucleotide
triphosphate of from 1:50 to 1:1000 (c) a polymerase enzyme which
incorporates dideoxynucleotides into an extending nucleic acid
polymer at a rate which is no less than 0.4 times the rate of
incorporation of deoxynucleotides.
4. The kit according to claim 3, wherein the mole ratio is from
1:100 to 1:500.
5. The kit according to claim 3, wherein at least one of the
primers is labeled with a fluorescent label.
6. The kit according to claim 3, wherein the primers are each
labeled with a spectroscopically-distinct fluorescent label.
7. The kit according to claim 3, wherein the target microorganism
is Chlamydia trachomatis.
8. The kit according to claim 7, wherein the first and second
primers are selected from the group consisting of the
oligonucleotides given by Seq. ID. Nos. 1-17.
9. The kit according to claim 3, wherein the target microorganism
is human immunodeficiency virus.
10. The kit according to claim 9, wherein the first and second
primers are selected from the group consisting of the
oligonucleotides given by Seq. ID. Nos. 18-20.
11. The kit according to claim 3, wherein the target microorganism
is human papilloma virus.
12. The kit according to claim 11, wherein the first and second
primers are selected from the group consisting of the
oligonucleotides given by Seq. ID. Nos. 21-22.
Description
[0001] This application is a continuation-in-part of pending U.S.
patent applications Ser. Nos. 09/009,483 filed Jan. 20, 1998,
08/640,672 filed May 1, 1996, now U.S. Pat. No. 5,789,168,
08/684,498 filed Jul. 19, 1996, now U.S. Pat. No. 5,830,657, and
08/807,138 filed Feb. 27, 1997, now U.S. Pat. No. 5,888,736, and of
International Patent Application No. PCT/US97/07134 filed Apr. 29,
1997 designating the United States, and published as International
Publication No. WO 97/41259 on Nov. 6, 1997, all of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This application relates to a method for detection and
identification of microorganisms, including in particular
pathogenic microorganisms, and to compositions and kits useful in
practicing the method. The invention can be applied to detection of
viruses, including HIV and hepatitis, bacteria, including
Chlamydia, fungi, including Cryptococcus neoformans and protozoa,
including Trypanosoma cruzi. This application also relates to DNA
sequencing reactions, and in particular to improved bi-directional
sequencing reaction protocols making use of thermally stable
polymerase enzymes.
[0003] Detection of the presence of pathogenic microorganisms
through DNA-based technology is emerging as an important tool in
the diagnosis of many diseases. For example, diagnosis of Chlamydia
trachomatis infections, the most common bacterial sexually
transmitted disease in North America, is shifting from traditional
methods such as culture, enzyme immunoassay (EIA) and direct
fluorescent antibodies (DFA) to DNA-hybridization diagnostics.
Roche Diagnostic Systems, Inc. (Nutley, N.J.) manufactures
Amplicor.TM., a test which detects C. trachomatis and Neisseria
gonorrohoeae by the hybridization of a pathogen specific probe to
PCR amplified products, detectable by a color change/optical
density technique. Abbott Laboratories (Abbott Park, Ill.) makes
UriProbe, also a test for C. trachomatis and N. gonorrohoeae, which
relies on the ligase chain reaction (LCR). The LCR method,
described in Patent Applications WO 9320227, WO 9300447, WO
9408047, WO 9403636, EP 477 972 uses thermostable ligase enzyme to
ligate two DNA probes which hybridize in ligatable juxtaposition on
a template DNA strand, thus generating a detectable ligated DNA
fragment only if the template DNA is present. A multiplex PCR assay
for C. trachomatis has also been described in Mahony et al., J.
Clin. Microbiol. 33: 3049-3053 (1995).
[0004] The ideal DNA sequencing procedure for use in a diagnostic
environment would have the following characteristics: (1) it would
be able to utilize a DNA-containing sample which had been subjected
to only minimal pretreatment to make the DNA accessible for
sequencing; (2) it would require combining this sample with only a
single reaction mixture, thus reducing risk of error and
contamination, and increasing the ease with which the procedure can
be automated; and (3) it would require a short amount of time to
perform the sequence determination, thus decreasing the marginal
costs in terms of equipment and labor for performing the test.
[0005] A wide variety if infectious pathogens that can be detected
by DNA-based methods are listed in Diagnostic Molecular
Microbiology, Persing et al., eds. American Society for
Microbiology, Washington D.C. (1993). This text details diagnostic
tests for bacteria, virus, fungi, and protozoa. Diagnostic tests
are also proposed for identifying the presence of drug resistance
genes or toxin genes.
[0006] Although these tests are generally effective for identifying
an infectious disease-causing organism if present, they do not
routinely provide information concerning the specific serotype,
variant or form of the infecting organism. Depending on the
organism in question, this information can be significant in
determining the likely course of the infection, for determining the
most appropriate therapeutic approach and for epidemiological
purposes. Furthermore, the previously known assays involve several
steps and are therefore more susceptible to systematic error than
would be a test with fewer steps. Thus, there remains a need for a
simple test format which is generally applicable to the detection
of microorganisms, including infectious disease-causing
microorganisms, and particularly for a simple test which provides
an indication of the specific nature, e.g., the serotype, of the
organism. It is an object of the present invention to provide such
a test.
[0007] It is an object of the present invention to provide reagent
combinations useful in performing tests for infectious
disease-causing microorganisms, including Chlamydia human papilloma
virus(HPV) and HIV.
[0008] It is a further object of the present invention to provide
kits useful in performing tests for infectious disease-causing
microorganisms, including Chlamydia, HPV and HIV.
[0009] It is an additional object of the present invention to
provide an improved method for bi-directional sequencing of DNA
samples which is well-suited for use in the diagnostic environment
and for automation.
[0010] It is an additional object of the invention to provide a
method for bi-directional sequencing of DNA which utilizes a
DNA-containing sample which has been subjected to only minimal
pretreatment to make the DNA accessible for sequencing.
[0011] It is still a further object of the invention to provide a
method for bi-directional sequencing of DNA which requires
combining a complex DNA-containing sample with only a single
reaction mixture, thus reducing risk of error and contamination,
and increasing the ease with which the procedure can be
automated.
SUMMARY OF THE INVENTION
[0012] The present invention provides a method for the evaluation
of a sample for the presence of a target microorganism which can be
performed directly on a natural abundance DNA preparation obtained
from the sample in a single reaction vessel. The method of the
invention comprises the steps of:
[0013] (a) combining the natural abundance DNA preparation with
first and second primers, a nucleotide triphosphate feedstock
mixture, a chain-terminating nucleotide triphosphate and a
thermally stable polymerase enzyme which incorporates
dideoxynucleotides into an extending nucleic acid polymer at a rate
which is no less than 0.4 times the rate of incorporation of
deoxynucleotides to form a reaction mixture, said first and second
primers binding to the sense and antisense strands of the DNA of
the target microorganism, respectively, and flanking a selected
region within the genome of the target microorganism;
[0014] (b) exposing the reaction mixture to a plurality of
temperature cycles each of which includes at least a high
temperature denaturation phase and a lower temperature extension
phase, thereby producing a plurality of species of terminated
fragments if DNA from the target microorganism is present in the
sample, each species of terminated fragment corresponding to a
different incorporation position for the chain-terminating
nucleotide triphosphate in the DNA of the target microorganism;
and
[0015] (c) evaluating the terminated fragments produced to
determine the incorporation positions of the chain-terminating
nucleotide triphosphate. Based on the incorporation positions, not
only the presence but also the specific nature, e.g. the serotype,
of any target microorganism present can be determined.
[0016] The present invention also provides a composition comprising
a mixture of four deoxynucleotide triphosphates and at least one
dideoxynucleotide triphosphate corresponding to one of the four
deoxynucleotide triphosphates, wherein the dideoxynucleotide
triphosphate is present in a mole ratio to the corresponding
deoxynucleotide triphosphate of from 1:50 to 1:500, said
composition further comprising a thermally stable polymerase enzyme
which incorporates dideoxynucleotides into an extending nucleic
acid polymer at a rate which is no less than 0.4 times the rate of
incorporation of deoxynucleotides.
[0017] Also provided is a kit for detection of a target
microorganism comprising, in packaged combination, the above
composition and a pair of primers which bind to the sense and
antisense strands, respectively, and flank a selected region within
the genome target microorganism.
[0018] The present invention also provides a method for
bidirectional sequencing a region of interest in a DNA sample. The
method can be carried out using a single set of reagents which is
added to a minimally-treated sample to produce useful sequencing
results.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The compositions and kits of the present invention can be
employed in a bi-directional DNA sequencing method in many contexts
including: (1) detection of mutations, particularly mutations of
medical significance, in samples derived from a human patient,
animal, plant or microorganism; (2) determination of HLA type
ancillary to transplant procedures; (3) detection and
identification of microorganisms, particularly pathogenic
microorganisms, in a sample; and (4) in-situ sequencing reactions
to produce sequencing fragments within a histological specimen
which are then removed from a selected location on the tissue
preparation and loaded onto a gel for sequence analysis. This
latter approach is particularly useful for evaluation of archived
samples in retrospective studies where the outcome of a disease
condition is known, but the potentially causative mutation is not.
This method can be used with labeled primers for single base
sequencing (or multiple-base sequencing using multiple tissue
samples).
[0020] In the context of detecting and identifying microorganisms,
the invention provides a novel approach to the evaluation of a
sample for the presence of a target microorganism and for the
identification of the specific nature of any organism found to be
present. The target microorganism may be virus, bacteria, fungi or
protozoa. Specific non-limiting examples of microorganisms to which
the invention can be suitably applied include bacteria such as
Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia
chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema
pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma
pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella
dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus
influenzae, Neisseria meningitidis, Streptococcus pneumonia, S.
agalactiae, and Listeria monocytogenes; viruses such as Human
Immunodeficiency Virus Type 1 (HIV-1), Human T-Cell Lymphotrophic
Virus Type 1 (HTLV-1), Hepatitis B Virus (HBV), Hepatitis C Virus
(HCV),Herpes Simplex, Herpesvirus 6, Herpesvirus 7, Epstein-Barr
Virus, Cytomegalovirus, Varicella-Zoster Virus, JC Virus,
Parvovirus B19, Influenza A, B and C, Rotavirus, Human Adenovirus,
Rubella Virus, Human Enteroviruses, Genital Human Papillomavirus
(HPV), and Hantavirus; fungi such as Cryptococcus neoformans,
Pneumocystis carinii, Histoplasma capsulatum, Blastomyces
dermatitidis, Coccidioides immitis, and Trichophyton rubrum; and
protozoa such as Trypanosoma cruzi, Leishmania sp., Plasmodium,
Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora
sp. and Eimeria sp. The method of the invention may also be used
for Cryptosporidium oocyst detection; for identification of
bacterial toxin genes, such as the toxin genes from Vibrio cholerae
01, enterotoxigenic Escherichia coli, Shigella sp., enteroinvasive
E. coli, Helicobacter pylori (formerly Campylobacter pylori),
toxigenic Clostridium difficile, Staphylococcus aureus, and
Streptococcus pyogenes exotoxins; and for identification of
anti-microbial resistance loci such as rifampin resistance
mutations in Mycobacterium tuberculosis and M. leprae; HIV Drug
Resistance, erm Erythromycin Resistance Genes,
methicillin-resistance genes in Staphylococcus,
Penicillinase-Producing genes in Neisseria gonorrhoeae, genes
encoding aminoglycoside-modifying enzymes, genes encoding an
extended spectrum of Beta-Lactamases, fluoroquinolone and isoniazid
resistance mutations in Mycobacterium tuberculosis, and genes
encoding vancomycin resistance in Enterococci.
[0021] The method of detecting and identifying the presence of a
target microorganism may utilize bi-directional DNA sequencing, in
which a natural abundance DNA-containing sample suspected to
contain the target microorganism is combined in a reaction mixture
with (1) first and second primers that hybridize with the sense and
antisense strands of the DNA of the target microorganism,
respectively, and flank a selected region within the genome of the
target microorganism, (2) a nucleotide triphosphate feedstock
mixture, (3) at least one chain-terminating nucleotide triphosphate
and (4) a polymerase enzyme which incorporates dideoxynucleotides
into an extending nucleic acid polymer at a rate which is no less
than 0.4 times the rate of incorporation of deoxynucleotides to
form a reaction mixture. This reaction mixture is processed through
a plurality of thermal cycles. Each thermal cycle includes at least
an extension step which is performed at a temperature of around 68
to 75.degree. C. and a denaturation step performed at a temperature
of around 90 to 98.degree. C. In addition, the thermal cycles may
include a separate annealing step performed at a temperature of 50
to 70.degree. C.
[0022] During each cycle, the primers each anneal to the respective
strand of any target DNA present in the sample, and primer chain
extension using the polymerase enzymes and the nucleotide
triphosphate feedstocks proceeds until terminated by incorporation
of a chain-terminating nucleotide triphosphate. This results in the
production of sequencing fragments comparable to those generated in
a conventional sequencing reaction. Analysis of these fragments
provides information concerning the sequence of the selected region
of the target DNA, and thus of the serotype of the target
microorganism. Those extension products which are not terminated
prior to reaching the region complementary to the other primer can
serve as template for generation of sequencing fragments in later
cycles, although this generally occurs to a very small extent. This
method also provides for simultaneously determining the positions
of a selected nucleotide base in a target region of both strands of
a denatured duplex nucleic acid polymer. The method of the
invention differs from the prior art, because the first and second
oligonucleotide primers are each labeled with different,
spectroscopically-distinguishabl- e fluorescent labels. The method
therefore obtains information about both DNA strands simultaneously
while providing improved sensitivity as a result of the non-linear
increase in the amount of DNA which results from the production of
additional templates molecules from unterminated fragments.
[0023] Among the advantages of the present invention is the ability
to perform an evaluation directly on a "natural abundance" DNA
sample. The nature of the initial sample will depend on the nature
of the target microorganism. For example, in the case of Chlamydia,
the initial sample employed in the present invention is suitably a
urine sample, genital scraping or genital swab taken from a human
patient, although other samples which are suspected of containing
Chlamydia can also be tested using the method of the invention.
Similarly, to test for HIV infection, the preferred sample is a
blood sample.
[0024] As used herein a "natural abundance sample" is a sample
which has been treated to make DNA in the sample accessible for
hybridization with oligonucleotide primers, for example by lysis,
centrifugation to remove cellular debris and proteolytic digestion
to expose the DNA, but which has not been subjected to a
preferential purification or amplification step to increase the
amount of target DNA relative to non-target DNA present in the
initial sample. The term "natural abundance" does not, however,
require the presence of all the DNA from the original sample. Thus,
a complex sample containing just nuclear DNA, or just mitochondrial
DNA or some subfraction of nuclear or mitochondrial DNA obtained by
isolation from a tissue sample but not subjected to preferential
amplification would be a "natural abundance" sample within the
meaning of that term in the specification and claims of this
application. The term "natural abundance" would also include a DNA
sample prepared by conversion, for example by reverse
transcription, of a total mRNA preparation or the genome of an RNA
virus to cDNA; DNA isolated from an individual bacterial colony
growing on a plate or from an enriched bacterial culture; and a
viral DNA preparation where substantially the entire viral genome
is isolated. The term "natural abundance" does not encompass a
sample in which the isolated DNA is not a complex combination of
DNA molecules, and thus would not encompass, for example, a
purified plasmid preparation containing only a single species of
plasmid.
[0025] Natural abundance samples of mammalian DNA can be prepared
from fluid samples, e.g., blood or urine or tissue samples by any
of a number of techniques, including lysis, centrifugation to
remove cellular debris and proteolytic digestion to expose the DNA;
salt precipitation or standard SDS-proteinase K-phenol extraction.
Natural abundance samples can also be prepared using kits, for
example the Gentra PURE GENE DNA Isolation Kit.
[0026] Primers used in the method of the present invention can be
any pair of primers which hybridize with the sense and antisense
strands DNA of the target microorganism flanking a selected region
of diagnostic relevance, and which do not both hybridize to
neighboring locations in human DNA or other microbial DNA
potentially found in the sample. As used herein, the term
"flanking" will be understood to mean the positioning of primers at
the 5'-ends of the selected region on each DNA strand, such that
extension of the primers leads to replication of the region between
the primers. The primers are preferably selected such that the
primer pair flanks a region that is about 500 bp or less, although
primers spanning larger regions of DNA can be utilized with
adjustments to the sequencing mixture (generally an increase in the
relative amount of deoxynucleotide triphosphates) to increase the
amount of longer sequencing fragments produced.
[0027] Primers can be selected to hybridize with highly conserved
regions which are the same in all variants of the target
microorganism or can be prepared as degenerate primers to take
known sequence variations at the primer site into account. Thus,
the first and second primers of the invention may each be a
discrete oligonucleotide species, or may be a set of
oligonucleotide primers with similar but not identical sequences.
Primers can also be selected to bind to the sense and antisense
strands of DNA flanking a region of the genome of the target
microorganism which is constant across all known variants and forms
of the microorganism, in which case the method of the invention
would provide detection but not any specific qualitative
characterization of the microorganisms, i.e., such primers could
not provide discrimination between subspecies, serovars, strains,
sub-types, biovars, variants, serotypes or between closely related
species of the target microorganism. An example of such a primer
pair is a primer pair that binds to the cryptic plasmid of C.
trachomatis which is recognized as a suitably specific target
sequence or detection purposes, but which is not known to vary from
strain to strain. Preferably, however, the primers employed will
flank a region of the target genome which is variable in sequence
depending on the serotype of the organism. Thus, for C. trachomatis
primers which flank portions of the omp1 gene are preferred.
Similarly, in the case of HIV detection, primers flanking known
mutation sites in the HIV protease gene or reverse transcriptase
gene produce fragments which permit both detection of HIV and the
identification of the HIV variant present in the sample. Primers
MY09 and MY11 (See example 10) give sequence information for most
relevant types of human papilloma virus (HPV) but not other
viruses.
[0028] In an alternative embodiment, primer pairs are selected
which, when treated under the conditions of the invention, give
sequence information from a much wider variety of organisms. This
is the case with eubacterial "universal" primers such as 91E and
13B listed in Appendix I which can be used to obtain sequence data
from the 16S rDNA gene of many bacteria. These primers are useful
for identifying which bacterium is present in a septic blood
culture, or any other pure but unknown culture. Patient samples
which contain a broad range of bacteria will give a complex result,
consisting of many overlapping sequences when tested with these
primers. The complex result may, in some cases, provide useful
information about the bacteria present. However, in the normal
course, it is advantageous to separate out the species, i.e. by
plating them out first. In this case, individual pure colonies can
be selected and identified.
[0029] In still another embodiment, the primer pairs are selected
to determine whether a specific gene is present in the patient
sample. The gene can be a toxin gene, a virulence gene, an
anti-biotic resistance gene or a specific mutation which confers
drug resistance or the like. Such a test can determine if a
micro-organism is present and if it carries the gene at the same
time.
[0030] Primers for other microorganisms can be derived from known
sequence information. Appendix I lists a collection of suitable
primer pairs for various other microorganisms which are taken from
Persing et al., supra.
[0031] One or both of the primers may be labeled with a detectable
label at the 5'-end thereof, particularly a fluorescent label such
as fluorescein or a cyanine dye such as Cy 5.5. If labels are used
on both primers, the labels selected should be
spectroscopically-distinct, i.e., they should have either a
different excitation spectrum or a different emission spectrum such
that one primer can be distinguished from the other. When both
primers are labeled with different detectable labels, the sequence
of both strands of the sample can be determined in a single
reaction.
[0032] The nucleotide triphosphate feedstock mixture is a standard
mixture of the four conventional bases (A, C, G and T) in a buffer
suitable for template-dependent primer extension with the enzyme
employed. As will be appreciated by persons skilled in the art, the
specific concentrations of the nucleotide triphosphates and the
nature of the buffer will vary depending on the enzyme employed.
Standard buffers and reagent concentrations for various known
polymerase enzymes may be employed in the invention.
[0033] The reaction mixture used in the present invention also
includes at least one type of chain-terminating nucleotide
triphosphate. Separate reactions for the four different types of
bases may be run either concurrently or successively. Running all
four bases concurrently comports with conventional sequencing
practice. However, a preferred embodiment of the present invention
combines the single vessel methodology of this application with
"single track sequencing" which is described in commonly assigned
U.S. Pat. No. 5,835,189. In single track sequencing, the
determination of the positions of only one (or in any event less
than 4) nucleotide(s) of a target sequence is frequently sufficient
to establish the presence of and determine the qualitative nature
of a target microorganism by providing a finger-print or bar-code
of the target sequence that may be sufficient to distinguish it
from all other known varieties of the sequence. Throughput is
increased by reducing the number of reactions and electrophoresis
runs required to identify a sequence. By selection of the order of
bases tested, and intermediate analysis, it may be unnecessary to
run all four bases to determine the presence and specific
qualitative nature of any target microorganism present in the
sample.
[0034] The polymerase enzyme used in the invention is a
thermostable polymerase enzyme which incorporates
dideoxynucleotides into an extending nucleic acid polymer at a rate
which is no less than 0.4 times the rate of incorporation of
deoxynucleotides. ThermoSequenase.TM. is exemplary of such an
enzyme. Reeve et al., Nature 376: 796-797 (1995). Tabor et al. have
also described enzymes which have increased processivity and
increased levels of incorporation of dideoxynucleotides. (See
EP-A1-0 655 506, which is incorporated herein by reference) Roche
sells an enzyme under the trademark TAQ-FS which meets these
criteria as well.
[0035] The absolute and relative amounts of nucleotide
triphosphates and chain-terminating nucleotide triphosphates may be
optimized for the particular enzyme employed. In general, however,
the nucleotide triphosphates will be included at in the reaction
mixture at concentrations of from 250 .mu.M to 1.5 mM, and the
chain-terminating nucleotide triphosphate will be included at a
level of from 0.5 .mu.M to 30 .mu.M to produce compositions in
which the mole ratio of the chain terminating nucleotide
triphosphate to the corresponding nucleotide triphosphate is from
1:50 to 1:1000, preferably from 1:100 to 1:500. This will result in
incorporation of a chain-terminating nucleotide triphosphate into
from 30 to 100 percent of the extending polymer chains formed
during the thermal cycling of the reaction mixture.
[0036] The method of the invention is suitably practiced using a
kit which provides the appropriate reagents in conveniently
packaged form. To reduce the number of sample preparation steps,
and thus to reduce the risk of erroneous results, such a kit will
suitably include at least one pre-prepared mixture comprising all
four nucleotide triphosphates and at least one chain terminating
nucleotide triphosphate, where the mole ratio of chain terminating
nucleotide to the corresponding deoxynucleotide triphosphate is
from 1:50 to 1 :1000, preferably 1:100 to 1:500.
[0037] The invention will now be further described by way of the
following non-limiting examples.
EXAMPLE 1
[0038] The presence of the sexually transmitted disease pathogen
Chlamydia trachomatis in a patient sample is detected according to
the method of the invention as follows.
[0039] Urine samples from patients suspected of carrying a sexually
transmitted disease pathogen are prepared for sequence- based
diagnosis as follows. 100 ul of first void urine are deposited in a
sterile microcentrifuge tube. The tube is centrifuged at
12,000.times.g for 20 min; the supernatant is removed. 100 ul of
Lysis Solution (Proteinase K@100 g/ml; 1% Tween 20) is added to the
bacterial pellet and incubated 1 h at 55.degree. C., or 18 h at
room temperature. After a final incubation at 95.degree. C. for 10
minutes, 200 ul of Geneclean II glass milk is added, according to
the manufacturer's instructions. (Bio 101, Inc) DNA is eluted in 10
ul of double distilled H.sub.2O. (A lysis solution control may be
prepared if desired, by adding the lysis solution to a sterile tube
(a tube without any urine pellet), and treating this tube like the
others.)
[0040] The sample natural abundance DNA is then treated according
to the method of the invention with a pair of primers and reagents
to identify the sequence of a C. trachomatis gene present in the
sample, if any. A suitable C. trachomatis specific target for
sequencing is the cryptic plasmid. Primers that may be used are
1 Name Sequence KL1: TCCGGAGCGA GTTACGAAGA [SEQ ID NO:1] KL2:
ATTCAATGCC CGGGATTGGT [SEQ ID NO:2]
[0041] These sequencing primers were employed previously for PCR
amplification reactions, but not sequencing (Mahony et al.,
"Confirmatory polymerase chain reaction testing for Chlamydia
trachomatis in first void urine from asymptomatic and symptomatic
men" J. Clin Microbiol. 30:2241-2245 (1992)).
[0042] Either primer may be labeled at the 5'-end with a detectable
label such as a Cy5.5 fluorophore. If both primers are labeled,
they should be distinguishable. Labels are selected on the basis of
the instrument employed for detection. Labeling reactions are
performed according to methods well known in the art, such as
amidite labeling or dye-ester condensation.
[0043] The sequencing reaction mixture is prepared by combining 2.5
ul of the prepared DNA sample, 0.67 ul of 10 uM primer KL1 (labeled
with Cy5.5), 0.45 ul of KL2 primer at 10 uM, 2 ul of
THERMOSEQUENASE reaction buffer (250 mM Tris-HCl pH 9.0@25.degree.
C., 39 mM MgCl.sub.2), 2 ul of THERMOSEQUENASE enzyme (Amersham
Life Sciences) diluted 1/10 in the dilution buffer provided with
the enzyme and 5.38 ul of double distilled H.sub.2O. The final
volume is 13 ul.
[0044] 3 ul of the sequencing reaction mixture is placed in each of
4 clean tubes and covered with one drop of mineral oil (Sigma
Chemical Co., Cat# M-5904). The tube is placed in a PTC-100 thermal
cycler (M.J. Research, Me.) and heated for 3 min at 94.degree. C.,
then cooled to 85.degree. C. One of the following termination
mixtures are then added to each of the 4 tubes:
[0045] 3 ul of dNTP:ddATP (1 mM each dNTP, 3.3 uM ddATP) in tube
A.
[0046] 3 ul of dNTP:ddCTP (1 mM each dNTP, 3.3 uM ddCTP) in tube
C.
[0047] 3 ul of dNTP:ddGTP (1 mM each dNTP, 3.3 uM ddGTP) in tube
G.
[0048] 3 ul of dNTP:ddTTP (1 mM each dNTP, 3.3 uM ddTTP) in tube
T.
[0049] The dNTP:ddNTP mixes are preferably heated to 85.degree. C
when added to the tube. The reaction mixture is mixed well and it
is subjected to the following thermal cycling regime for 55
cycles:
[0050] 94.degree. C./30 sec.
[0051] 60.degree. C./30 sec.
[0052] 70.degree. C./1 min.
[0053] After the last cycle, the tubes are kept at 70.degree. C.
for 2 min, then cooled to 4.degree. C. until ready for loading. To
view the reaction products, 6 ul of loading buffer (dye/stop
solution) is added to each tube. The aqueous phase (the bottom
phase disposed under the oil layer) is removed and put it in
another tube. The sample is heated to 75.degree. C. for 3 min, and
put on ice. 2 ul of each sample is loaded in each well of a
MicroGene Blaster automated DNA sequencer (Visible Genetics Inc.,
Toronto, ON). The reaction products are electrophoretically
separated and detected. The data is analyzed using GeneObjects
software (Visible Genetics Inc., Toronto, ON) to base-call (i.e.
determine the DNA sequence) of the samples. The base-called
sequence is compared to the known C. trachomatis sequence to
confirm diagnosis. Results are reported to the patient file.
EXAMPLE 2
[0054] The method of the invention may be employed to identify not
only the presence of C. trachomatis in a patient sample but also
the strain identity. Health care workers currently seek to
distinguish among Chlamydia trachomatis strains to determine the
molecular epidemiologic association of a range of diseases with
infecting genotype (See Dean, D. et al "Major Outer Membrane
Protein Variants of Chlamydia trachomatis Are Associated with
Severe Upper Genital Tract Infections and Histopathology in San
Francisco." J. Infect. Dis. 172:1013-22 (1995)).
[0055] A suitable strain specific target for C. trachomatis is the
omp1 (outer membrane protein) gene which has at least 4 variable
sequence ("VS") domains that may be used to distinguish among the
15 known genotypes of C. trachomatis (Yuan, Y et al. "Nucleotide
and Deduced Amino Acid Sequences for the Four Variable Domains of
the Major Outer Membrane Proteins of the 15 Chlamydia trachomatis
Serovars" Infect. Immun. 57 1040-1049 (1989)).
[0056] Strain identification is achieved using the method of
Example 1 with the following modifications. First of all, because
of the length of the VS domains, separate reactions are performed
to obtain sequence from VS1/VS2 and VS3/VS4. The following
oligonucleotide primers may be employed:
2 Name Sequence For VS1/VS2: MF21 CCGACCGCGT CTTGAAAACA GATGT [SEQ.
ID NO. 3] MB22 CACCCACATT CCCAGAGAGC T [SEQ. ID NO. 4] For VS3/VS4
MVF3 CGTGCAGCTT TGTGGGAATG T [SEQ. ID NO. 5] MB4 CTAGATTTCA
TCTTGTTCAA TTGC [SEQ. ID NO. 6]
[0057] These sequencing primers were employed previously for PCR
amplification reactions, but not sequencing. Mahoney et al.,
supra.
[0058] These oligonucleotide primers are used in separate reactions
in place of KL1 and KL2 in Example 1. The sample preparation and
sequencing reactions are performed as in Example 1. The reaction
products are electrophoretically separated and detected on a
MicroGene Blaster automated DNA sequencing apparatus (Visible
Genetics Inc., Toronto, ON). The data is analyzed using GeneObjects
software to base-call the samples and to compare the data to the
known varieties of C. trachomatis. Pure populations generally give
unambiguous sequence data. Where heterozygous mixed populations are
detected, a circumstance thought to occur in 1-3% of clinical C.
trachomatis samples, the software identifies the strains which
could be combined to result in the particular heterozygote sample
detected.
EXAMPLE 3
[0059] Strain-specific C. trachomatis identification over the
VS1/VS2 domain can be achieved according to the method in Example
1, by using the following degenerate primers sets:
3 Forward OMP291: AGCATGCGTR TKGGTTACTA YGG [SEQ ID NO. 7] (labeled
with Cy5.5). Base 175 to 197 of the ORF of the omp 1 gene of C.
trachomatis. Forward OMP314A: TGACTTTGTT TTCGACCGYG TTTT [SEQ ID
NO. 8] (labeled with Cy5.5). Base 198 to 221 of the ORF of the omp
1 gene of C. trachomatis. Reverse OMP722: CTAAAGTYGC RCATCCACAT TCC
[SEQ ID NO. 9] Base 637 to 615 of the ORF of the omp 1 (in serovar
K) gene of C. trachomatis. The primer may not have the EXACT SAME
sequence as in serovar K. Reverse OMP71 1: CATCCACATT CCCASARAGC
TGC [SEQ ID NO. 10] Base 626 to 604 of the ORF of the omp 1 (in
serovar K) gene of C. trachomatis. The primer may not have the
exact same sequence as in serovar K.
[0060] Base 637 to 615 of the ORF of the omp1 (in serovar K) gene
of C. trachomatis. The primer may not have the EXACT SAME sequence
as in serovar K.
[0061] Reverse
[0062] OMP711: CATCCACATT CCCASARAGC TCC [SEQ ID NO. 10]
[0063] Base 626 to 604 of the ORF of the omp1 (in serovar K) gene
of C. trachomatis. The primer may not have the exact same sequence
as in serovar K.
[0064] These primers sets are preferably used in the following
combinations:
[0065] (1) OMP291 -MP722, sequencing a 455 to 463-bp (depending on
the serotype) fragment of the omp1 gene of C. trachomatis; or
[0066] (2) OMP314A-OMP711, sequencing a 421 to 430-bp (depending on
the serotype) fragment of the omp1 gene of C. trachomatis.
EXAMPLE 4
[0067] The method as exemplified in Examples 1, 2 and 3 may be
further improved by employing different labels, preferably
fluorescent labels, on the different primers for use in a multi-dye
sequencer. This method takes advantage of the fact that a given
termination mixture containing, for example, ddATP will give chain
termination products for the A nucleotide in both directions. The
different primer labels means that one reaction mixture loaded in a
single lane of an automated DNA sequencing apparatus designed to
detect the two labels (a "multi-dye sequencer") will identify the A
nucleotide of both sense and antisense strands. Separate reactions
are performed for the other 3 nucleotides. Using only 4 lanes of an
electrophoresis gel, and 4 reaction mixtures, the DNA sequences of
both the sense and anti-sense strands can be obtained. This
information allows the operator to resolve any ambiguities that may
be present.
[0068] Use of two different labels lends itself to a further
improvement. As noted above, in a reaction according to the
invention, the results of the ddATP reaction will give chain
termination products for the A nucleotide in both directions, Since
the A nucleotide in one direction corresponds to the T nucleotide
in the other, a single reaction can provide the location of two
bases. A second termination reaction with, for example, ddCTP will
then obtain the positions of the other two nucleotides, C and G.
Thus only two lanes of an electrophoresis gel and 2 reaction
mixtures are required to identify the location of all 4 bases of
the sequence.
[0069] A suitable multi-dye sequencer for use with this aspect of
the invention, is the Applied Biosystems 377 Prism automated DNA
sequencer (Applied Biosystems Inc., Foster City, Calif.). The
fluorescent labels are selected to be detectable on the 377
instrument. Instead of the dye-terminator chemistry suggested in
the Applied Biosystems product literature, however, the fluorescent
labels must be conjugated to the 5' end of the primer molecules.
The samples are electrophoresed, detected and the detected data is
recorded.
[0070] Sophisticated software such as GeneObjects software (Visible
Genetics Inc, Toronto, Calif.) may be used to assist in evaluation
of the results. This software may employ the methods of commonly
assigned U.S. Pat. No. 5,853,979, U.S. patent application Ser. No.
08/670,534 and International Patent Application No. PCT/US96/11130,
published as WO97/02488, all of which are incorporated herein by
reference. In one of the methods, the single nucleotide data tracks
are evaluated and nucleotides are positioned relative to the known
(or standard) DNA sequence expected from the sample. When data
tracks are generated for each of the four nucleotides, the full DNA
sequence of the sample may be base-called. The base-called sequence
is then compared to the library of known sequences to determine
which C. trachomatis strain or strains are present in the
sample.
EXAMPLE 5
[0071] The sequence of both the sense strand and antisense strand
of a C. trachomatis cryptic plasmid gene may be obtained in a one
step reaction using the primers:
4 Concentration Amount Patient Sample DNA 11.25 ul KL1*Cy5.5 Primer
10 uM 3 ul CT1590*Fluoresceine Primer 10 uM 2 ul Enzyme Diluent
(Amersham plc) 8 ul ThermoSequenase Enzyme 32 U/ul 0.9 ul double
distilled H.sub.2O 24.2 ul
[0072] Combine the following materials and mix well:
5 Name Sequence KL1: TCCGGAGCGA GTTACGAAGA [SEQ ID NO. 1] CT1590:
ATGCCCGGGA TTGGTTGATC [SEQ ID NO. 11]
[0073] Take 11 ul of the mixture and add 2 ul of 13.times.buffer
[Tris-HCl 260 mM pH 8.3, MgCl.sub.2 39 mM] (final concentration 20
mM Tris-HCl pH 8.3, 3 mM MgCl.sub.2). Mix well and place 3 ul into
each of 4 tubes. Heat tube to 94.degree. C. for 5 mins then reduce
temperature to 85.degree. C. Add and mix 3 ul of an 85 C dNTP/ddNTP
solution consisting of 0.75 mM each dNTP and 2.5 uM of a chain
terminating nucleotide triphosphate (ddNTP) (use a different ddNTP
in each of the 4 tubes).
[0074] Treat the mixture to 60 cycles of the following thermal
cycling reactions: 94.degree. C. for 10 sec, 62.degree. C. for 15
sec, 70.degree. C. for 1 min. Upon completion, treat the mixture
for a final 5 min at 70 C. and then store at 4.degree. C. until
ready for loading. For viewing the reaction products, add an equal
volume of stop/loading solution (95% formamide plus a colored dye).
Take 1.5 ul and load in a single lane of a MicroGene Blaster
automated DNA sequencer (Visible Genetics Inc., Toronto). Load the
remaining mixture (@10.5 ul) in a single lane of an ALF Automated
Sequencer (Pharmacia LKB, Uppsala, Sweden). The reaction products
from the Cy5.5 labeled primer are detected on the MicroGene Blaster
using GeneObjects Software. The reaction products from the
fluorescein labeled primer are detected on the ALF Automated
Sequencer using GeneObjects Software. The base-calling results of
the Cy5.5 labeled primer were compared to the known sequence of the
gene by the GeneLibrarian component of GeneObjects.
EXAMPLE 6
[0075] As described in U.S. Pat. No. 5,834,189, not all 4
nucleotides of C. trachomatis, or any polymorphic or multiple
allelic locus of any gene or organism necessarily need to be
determined in order to ascertain which allele or variant is
present. In many cases, positioning less than four nucleotides may
be sufficient to determine with certainty which allele is present.
The method of Examples 1-4 may be modified to obtain single
nucleotide data tracks (or fragment patterns) by performing only
one of the termination reactions at a time.
[0076] In the case of detection and serotyping of C. trachomatis,
the evaluation of the A track alone over the first 100 nucleotides
of the omp1 gene, aligning to nucleotides 249-349 of the serovars C
and K, can distinguish the serovars. Appendix II is a text file
representation of the omp1 gene in each of the serovars. The
sequences are all aligned to the last (3') nucleotide of the
delectably labeled primer omp3l4A. (Appendix II shows sequences
starting 29 bp downstream of the 3'-nucleotide.) This illustration
differs from a traditional "consensus" sequence illustrations in
that all missing bases (usually represented by N's or raised
dashes) are deleted. The A's are illustrated in the order and
positions in which they would be expected to appear after a
sequencing reaction and upon detection by an automated DNA
electrophoresis apparatus.
[0077] If, in another microorganism, the A lane (or other preferred
first lane) were not sufficient to distinguish all types, a second
reaction for the C, G or T nucleotide could be performed to further
define the qualitative nature of any target microorganism present
in the sample. Because the sequences of the types are previously
known, the operator can determine which of the nucleotides provide
the greatest information and will analyze those nucleotides
first.
EXAMPLE 7
[0078] The presence of and strain identity of C. trachomatis in a
patient sample may be determined according to the methods of the
previous examples by substituting the following primer pairs. These
primers are used to determine the sequence of the omp1 gene
(publicly available at DNASIS Accession No. X62921).
[0079] Forward Primer (5' Primer) labeled with a detectable label
such as Cy5.5:
[0080] Primer OMP312: GGAGACTTTG TTTTCGACCG [SEQ ID NO 12]
[0081] Position 312-331 of X62921
[0082] and one of the following Reverse Primers (3' Primer)
(optionally labeled with a detectable label different from the 5'
primer):
6 Primer OMP708: CATTCCCACA AAGCTGCGCG [SEQ ID NO 13] Position
727-708 of X62921 Primer OMP706: TTCCCACAAA GCTGCGCGAG [SEQ ID NO
14] Position 725-706 of X62921 Primer OMP704: CCCACAAAGC TGCGCGAGCG
[SEQ ID NO 15] Position 723-704 of X62921
[0083] The following combination can be used to obtain DNA sequence
over the following maximum lengths:
[0084] OMP312-OMP708: 416-nt region of omp1
[0085] OMP312-OMP706: 414-nt region of omp1
[0086] OMP312-OMP704: 412-nt region of omp1
EXAMPLE 8
[0087] The presence of and strain identity of C. trachomatis in a
patient sample may be determined according to the method of
previous examples, using C. trachomatis ribosomal DNA (rDNA)
specific primers such as
7 CT220 ACCTTTCGGT TGAGGGAGAG TCTA [SEQ ID NO 16] and CT447
GGACCAATTC TTATTCCCAA GCGA [SEQ ID NO 17] Haydock et al., Chap 1.10
in Persing et al., supra.
EXAMPLE 9
[0088] The sequence of both the sense strand and antisense strand
of the protease gene of HIV-1 integrated into natural abundance DNA
of lymphocytes may be obtained in a one step reaction as
follows.
[0089] Natural abundance DNA is prepared from the patient blood
lymphocyte sample according to a standard method such as a standard
salting-out procedure (as provided by the Puregene DNA Isolation
Kit, Gentra Systems, Inc., Minneapolis) or by detergent and
proteinase K treatment (Current Protocols in Molecular Biology,
Eds. Ausubel, F. M. et al, (John Wiley & Sons; 1995)).
[0090] Combine the following materials and mix well:
8 Concentration Amount Patient Sample DNA 11.25 ul PR211F*Cy5.5
Primer 10 uM 3 ul or PR281*Cy5.5 Primer 10 uM 3 ul
PR526*Fluorescein Primer 10 uM 2 ul Enzyme Diluent (Amersham plc) 8
ul THERMOSEQUENASE Enzyme 32 U/ul 0.9 ul double distilled H.sub.2O
24.2 ul
[0091] The primers have the following sequences:
9 Choice of Forward Primers Name Sequence PR211F ATCACTCTTT
GGCAACGACC or [SEQ ID No. 18] (FORWARD), BASE 6 TO 26 OF THE
PROTEASE GENE PR281 CAGGAGCAGA TGATACAGTA TTAG [SEQ ID No. 19]
[0092] PR211F-PR526 creates a sequencing fragment of maximum size
340 bp. PR281-PR526 creates a sequencing fragment of maximum size
270 bp. Both regions contain the sequence of the various codons
where mutations are involved in protease inhibitor resistance
(Codons 46, 48, 54, 63 82 84 and 90).
[0093] Take 11 ul of the mixture and add 2 ul of 13.times.buffer
[Tris-HCl 260 mM pH 8.3, MgCl.sub.2 39 mM] (final concentration 20
mM Tris-HCl pH 8.3, 3 mM MgCl.sub.2). Mix well and place 3 ul into
each of 4 tubes. Heat tube to 94 C. for 5 mins then reduce
temperature to 85 C. Add and mix 3 ul of an 85 C. dNTP/ddNTP
solution consisting of 0.75 mM each dNTP and 2.5 uM of a chain
terminating nucleotide triphosphate (ddNTP) (use a different ddNTP
in each of the 4 tubes).
[0094] Treat the mixture to 60 cycles of the following thermal
cycling reactions: 94 C for 10 sec, 62 C. for 15 sec, 70 C. for 1
min. Upon completion, treat the mixture for a final 5 min at 70 C.
and then store at 4 C. until ready for loading. For viewing the
reaction products, add an equal volume of stop/loading solution
(95% formamide plus a coloured dye). Take 1.5 ul and load in a
single lane of a MicroGene Blaster automated DNA sequencer (Visible
Genetics Inc., Toronto). Load the remaining mixture (@10.5 ul) in a
single lane of an ALF Automated Sequencer (Pharmacia LKB, Uppsala,
Sweden). The reaction products from the Cy5.5 labelled primer are
detected on the MicroGene Blaster using GeneObjects Software. The
reaction products from the fluorescein labeled primer are detected
on the ALF Automated Sequencer using GeneObjects Software. The
base-called results from each primer were compared to the known
sequences of HIV-1 by GeneLibrarian (a component of GeneObjects
(Visible Genetics Inc, Toronto).
EXAMPLE 10
[0095] The presence and type of human papilloma virus (HPV) present
in a patient sample can be determined according to the method of
the invention by following the protocol in Example 1 with the
following modifications.
[0096] Patient sample DNA is extracted from 250 ul urine specimens
using Geneclean II (Bio 101, Inc.). The sample is then treated as
described previously but employing the degenerate primer pair:
10 Forward Primer: MY11 GCMCAGGGWC ATAAYAATGG [SEQ ID No. 21]
Reverse Primer: MY09 CGTCCMAARG GAWACTGATC [SEQ ID No. 22]
[0097] The reactions are performed as before, using ThermoSequenase
enzyme or the like.
[0098] Reaction products are detected on an automated
electrophoresis/detection device such as the MicroGene Blaster. The
sequence is analyzed and compared to the known varieties of HPV to
identify the type. The result is reported to the patient file.
[0099] Although the preferred embodiments of the invention has been
described above in some detail, it should be appreciated that a
variety of embodiments will be readily apparent to one skilled in
the art. The description of the invention is not intended to be
limiting to this invention, but is merely illustrative of the
preferred embodiments.
Sequence CWU 1
1
* * * * *