U.S. patent application number 10/045621 was filed with the patent office on 2003-05-01 for polymerase extension at 3' terminus of pna-dna chimera.
This patent application is currently assigned to PE Corporation (NY). Invention is credited to Chen, Caifu, Egholm, Michael.
Application Number | 20030082558 10/045621 |
Document ID | / |
Family ID | 23474127 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082558 |
Kind Code |
A1 |
Egholm, Michael ; et
al. |
May 1, 2003 |
Polymerase extension at 3' terminus of PNA-DNA chimera
Abstract
The invention provides methods and a kit for primer extension of
PNA-DNA chimera from template nucleic acids using polymerases,
nucleotide 5'-triphosphates, and primer extension reagents.
Structural requirements of the chimera for primer extension include
5 to 15 contiguous PNA monomer units, 3 or more contiguous
nucleotides, and a 3' hydroxyl terminus. The chimera and/or a
nucleotide is labelled with fluorescent dyes or other labels. The
methods include DINA sequencing, DNA fragment analysis, reverse
transcription, mini-sequencing, chromosome labelling,
amplification, and single nucleotide polymorphism (SNP)
detection.
Inventors: |
Egholm, Michael;
(Woodbridge, CT) ; Chen, Caifu; (Palo Alto,
CA) |
Correspondence
Address: |
PATTI SELAN, PATENT ADMINISTRATOR
APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
PE Corporation (NY)
850 Lincoln Centre Drive
Foster City
CA
94404
|
Family ID: |
23474127 |
Appl. No.: |
10/045621 |
Filed: |
October 24, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10045621 |
Oct 24, 2001 |
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09373845 |
Aug 13, 1999 |
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6316230 |
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Current U.S.
Class: |
435/6.11 ;
435/6.1; 435/69.1; 435/91.2 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 2537/143 20130101; C12Q 2525/107 20130101; C12Q 2525/107
20130101; C12Q 2525/107 20130101; C12Q 1/6869 20130101; C12Q 1/6858
20130101; C12Q 1/6853 20130101; C12Q 1/6858 20130101; C12Q 1/6853
20130101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
021/06; C12P 019/34 |
Claims
We claim:
1. A method of producing a non-radioisotopically labelled chimeric
extension product comprising the step of enzymatically extending a
PNA-DNA chimera in the presence of a template nucleic acid, a
polymerase and a primer extension reagent, wherein said primer
extension reagent comprises a nucleotide 5'-triphosphate capable of
effecting enzymatic chimera primer extension, and wherein the
chimera or the nucleotide 5'-triphosphate is labelled with a
non-radioisotopic label.
2. The method of claim 1 in which the PNA-DNA chimera has the
structure:P.sub.x-L-N.sub.y3'wherein: each P is independently a PNA
monomer; x is an integer from 5 to 15; L represents a covalent
linkage between P and N; each N is independently a nucleotide; and
y is an integer from 3 to 15; with the proviso that the 3' terminal
N has a 3' hydroxyl group.
3. The method of claim 1 in which the primer extension reagent
comprises a mixture of nucleotide 5'-triphosphates capable of
effecting continuous primer extension.
4. The method of claim 3 in which the mixture comprises four
different nucleotide 5'-triphosphates, and further wherein a
nucleotide 5'-triphosphate is ATP, dATP, 7-deaza dATP, 2-amino-ATP
or 2-amino-dATP, and another nucleotide 5'-triphosphate is GTP,
dGTP, or 7-deaza dGTP, another nucleotide 5'-triphosphate is CTP,
dCTP, 5-methyl-CTP, 5-methyl-dCTP or 5-propynyl dCTP, and another
nucleotide 5'-triphosphate is UTP, dUTP, dTTP, 5-Br-UTP, 5-Br-dUTP,
5-F-UTP, 5-F-dUTP or 5-propynyl-dUTP.
5. The method of claim 4 in which the mixture further includes a
terminating nucleotide 5'-triphosphate.
6. The method of claim 1 in which the nucleotide 5'-triphosphate is
a terminating nucleotide 5'-triphosphate.
7. The method of claim 5 or 6 in which the terminating nucleotide
5'-triphosphate is detectably labelled with a fluorescent dye.
8. The method of claim 1 in which the primer extension reagent
further comprises a mixture of terminating nucleotide
5'-triphosphates.
9. The method of claim 8 in which the mixture comprises four
different terminating nucleotide 5'-triphosphates, and further
wherein a terminating nucleotide 5'-triphosphate is ddATP, 7-deaza
ddATP, or 2',3'-dideoxy-dehydro-ATP, another terminating nucleotide
5'-triphosphate is ddGTP, 7-deaza ddGTP or
2',3'-dideoxy-dehydro-GTP, another terminating nucleotide
5'-triphosphate is ddCTP or 2',3'-dideoxy-dehydro-CTP, and another
terminating nucleotide 5'-triphosphate is ddTTP, ddUTP,
2',3'-dideoxy-dehydro-TTP or 2',3'-dideoxy-dehydro-UTP.
10. The method of claim 9 wherein each different terminating
nucleotide 5'-triphosphate is labelled with a different detectable
label.
11. The method of claim 2 wherein P.sub.x is a 2-aminoethylglycine
peptide nucleic acid.
12. The method of claim 2 in which each N is independently a
2'-deoxyribonucleotide.
13. The method of claim 2 in which each N is independently a
ribonucleotide.
14. The method of claim 2 wherein the nucleobases of N are selected
from the group consisting of C-5-alkyl pyrimidine,
2.6-diaminopurine, 2-thiopyrimidine, C-5-propyne pyrimidine,
phenoxazine, 7-deazapurine, isocytidine, pseudo-isocytidine,
isoguanosine, hypoxanthine, 8-oxopurine, and 4(3 H)-pyrimidone.
15. The method of claim 2 wherein the sugars of N are selected from
the group consisting of 29-O-alkyl-ribonucleotides,
2'-O-methyl-ribonucleotid- es, 2'-O-allyl-ribonucleotides, 2'-allyl
ribonucleotides, 2'-halo-ribonucleotides,
2'-O-methoxyethyl-ribonucleotides, 4'-.alpha.-anomeric nucleotides,
1'-.alpha.-anomeric nucleotides, 2',4'-linked nucleotides, and
bicyclic nucleotides.
16. The method of claim 1 wherein the PNA-DNA chimera is labelled
at the amino terminus of the PNA moiety.
17. The method of claim 1 wherein the nucleotide 5'-triphosphate is
labelled at the nucleobase.
18. The method of claim 17 wherein the nucleobase label sites are
the N-9 or C-8 positions of the purine or deazapurine, and the C-5
position of the pyrimidine.
19. The method of claim 1 wherein the label is selected from the
group consisting of fluorescent dyes, fluorescence quenchers,
hybridization-stabilizers, energy-transfer dye sets,
electrophoretic mobility modifiers, chemiluminescent dyes, amino
acids, proteins, peptides, enzymes, and affinity ligands.
20. The method of claim 19 where the fluorescent dyes are selected
from the group consisting of FAM, TET, HEX, JOE, TAMPA, d-TAMRA,
JODA, ROX, VIC, NED, dJON, dR139, 4,7-dichloro-fluoresceins,
4,7-dichloro-rhodamines- , and cyanines.
21. The method of claim 19 where the fluorescence quenchers are
selected from the group consisting of TAMRA, d-TAMRA, ROX, DABCYL,
DABSYL, malachite green, NTB, and cyanines.
22. The method of claim 19 where the hybridization-stabilizers are
minor groove binders.
23. The method of claim 19 where the minor groove binders are
selected from the group consisting of Hoechst 33258, CDPI.sub.1-3 ,
MGB1, netropsin, and distamycin.
24. The method of claim 19 where the affinity ligands are selected
from the group consisting of biotin, 2.4-dinitrophenyl,
digoxigenin, cholesterol, polyethyleneoxy, peptides, and
fluorescein.
25. The method of claim 2 wherein L is selected from the group
consisting of a covalent bond, alkyldiyl consisting of 1-20 carbon
atoms, aryldiyl, 0 linker, and --(CH.sub.2CH.sub.2O).sub.m-- where
m is 1 to 6.
26. The method of claim 1 in which the template nucleic acid is a
DNA and the polymerase is selected from the group consisting of
Klenow, T4, Bst, AmpliTaq, AmpliTaq Gold, AmpliTaq Stoffel
fragment, Sequenase, Vent, Pfu, and bacteriophage T7.
27. The method of claim 1 in which the template nucleic acid is an
RNA and the polymerase is a reverse transcriptase.
28. The method of claim 1 in which the template nucleic acid is a
metaphase or interphase chromosome.
29. The method of claim 28 in which the chromosome is
denatured.
30. The method of claim 1 in which the PNA-DNA chimera is
immobilized on a solid substrate.
31. The method of claim 30 in which the chimera is covalently
attached to the solid substrate, optionally with the aid of a
linker.
32. The method of claim 1 in which the template nucleic acid is
immobilized on a solid substrate.
33. The method of claim 32 in which the template nucleic acid is
covalently attached to the solid substrate, optionally with the aid
of a linker.
34. The method of claim 30 or 32 wherein the solid substrate is
selected from the group consisting of polystyrene,
controlled-pore-glass, silica gel, silica, polyacrylamide, magnetic
beads, polyacrylate, hydroxyethylmethacrylate, polyamide,
polyethylene, polyethyleneoxy, and copolymers and grafts of any of
the above solid substrates.
35. The method of claim 30 or 32 wherein the solid substrate is
selected from the group consisting of small particles, beads,
membranes, frits, slides, plates, micromachined chips,
alkanethiol-gold layers, non-porous surfaces, and
polynucleotide-immobilizing media.
36. A kit for primer extension comprising: a PNA-DNA chimera
primer, said primer comprising 5 to 15 contiguous PNA monomer
units, 3 to 15 contiguous nucleotides, and a 3' hydroxyl terminus;
one or more nucleotide 5'-triphosphates and; a polymerase enzyme,
wherein the chimera primer or a nucleotide 5'-triphosphate is
non-radioisotopically labelled.
37. The kit of claim 36 further comprising a template nucleic acid
comprising a sequence complementary to the chimera primer or
containing one or more mismatches to the chimera primer.
38. A method of sequencing a template nucleic acid, comprising the
steps of: a. generating a labelled primer extension product by
enzymatically extending a primer-template nucleic acid hybrid in
the presence of a polymerase and a terminating nucleotide
5'-triphosphate, wherein said primer is a PNA-DNA chimera and
either said primer or said terminating nucleotide 5'-triphosphate
is detectably and non-radioisotopically labelled; b. separating the
labelled primer extension products based on size; and c.
determining the sequence of the template nucleic acid.
39. The method of claim 38 wherein a nested set of labelled primer
extension products are generated by a mixture of enzymatically
extendable nucleotide 5'-triphosphates capable of supporting
continuous primer extension.
40. The method of claim 38 wherein the PNA-DNA chimera is
labelled.
41. The method of claim 38 wherein the terminating nucleotide
5'-triphosphate is labelled.
42. The method of claim 41 wherein the labelled, terminating
nucleotide 5'-triphosphate is selected from the group consisting of
a labelled ddNTP, a labelled 2'-amino, 2'-deoxynucleotide, a
labelled 2'-halo, 2'-deoxynucleotide, and a labelled 2',3'--
dideoxy-dehydronucleotide.
43. A method of reverse transcription comprising the step of
generating labelled primer extension products by enzymatically
extending a primer-template RNA hybrid in the presence of a reverse
transcriptase, a mixture of enzymatically-extendable nucleotide
5'-triphosphates capable of supporting continuous primer extension,
wherein said primer is a PNA-DNA chimera and either said primer or
a nucleotide 5'-triphosphate is non-radioisotopically labelled.
44. A method of DNA amplification comprising the steps of: a.
generating labelled amplification products by enzymatically
extending a primer-template nucleic acid hybrid in the presence of
two primers each of which is capable of hybridizing to the template
and wherein one or both of which is a PNA-DNA chimera primer, a DNA
polymerase and a mixture of enzymatically-extendable nucleotide
5'-triphosphates capable of supporting continuous primer extension,
wherein either said primers or nucleotide 5'-triphosphates are
non-radioisotopically labelled: and b. cycling the temperature to
effect denaturation, annealing, and primer extension to form an
amplification product by extension of the primers with nucleotide
5'-triphosphates; wherein one or both of the 5' terminii of the
amplification product bears the PNA sequence of the chimera
primers.
45. The method of claim 44 in which the amplification product is
immobilized by hybridization on a solid substrate comprising a
nucleic acid with a sequence complementary to the PNA sequence of
the amplification product.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 09/373,845,
filed Aug. 13, 1999, which claims priority under 35 U.S.C. 120
which is incorporated by reference.
I. FIELD OF THE INVENTION
[0002] The invention relates generally to the fields of enzymology
and nucleic acid analogs. Specifically, this invention is directed
to template-dependent, primer extension of PNA-DNA chimera using
polymerase enzyme and nucleotide 5'-triphosphates.
II. BACKGROUND
[0003] One of the most powerful and versatile tools available to
molecular biologists is the in vitro replication of nucleic acid
sequences by primer extension, as exemplified by the ubiquitous
techniques of polymerase chain reaction (PCR) (Mullis, 1987) and
DNA sequencing (Sanger, 1977). Both techniques include the steps
of: 1) hybridizing a short, e.g. 15-30 nt, synthetic
oligonucleotide primer to a single-stranded template nucleic acid;
and 2) enzymatically extending from the 3' hydroxyl terminus of the
primer in the presence of nucleotide 5'-triphosphates,
complementary to the template strand, and a polymerizing enzyme. By
this general primer extension method, sequencing information is
generated, template nucleic acids are amplified or copied, and
other genetic analysis tests are conducted. Results are optimized
through the choice and concentrations of primers, multiple primers,
enzymes, nucleotides, and other reagents, and the selection of
temperature, temperature cycling conditions, and other experimental
conditions.
[0004] The choice of primers has been primarily limited to
2'-deoxyoligonucleotide primers made by the phosphoramidite
chemistry method (Caruthers, 1983) on automated synthesizers
(Caruthers, 1984). Whereas nucleic acid analogs are known which
efficiently hybridize to DNA or RNA, some with comparable or
superior hybridization specificity and/or affinity, enzyme-mediated
formation of a new phosphodiester bond only occurs between a primer
having a 3' terminal hydroxyl and a nucleotide having a
5'-triphosphate, or a closely related isostere, i.e.
.alpha.-thiotriphosphate, etc. Most structural permutations in
either the primer or the nucleotide severely compromise the
efficiency of primer extension, or negate it totally.
[0005] Nucleic acid analogs are structural analogs of DNA and RNA
and which are designed to hybridize to complementary nucleic acid
sequences. Through modification the internucleotide linkage, the
sugar, and/or the nucleobase, nucleic acid analogs may attain any
or all of the following desired properties: 1) optimized
hybridization specificity or affinity, 2) nuclease resistance. 3)
chemical stability, 4) solubility, 5) membrane-permeability, and 6)
ease or low costs of synthesis and purification.
[0006] A useful and accessible class of nucleic acid analogs is the
family of peptide nucleic acids (PNA) in which the sugar/phosphate
backbone of DNA or RNA has been replaced with acyclic, achiral, and
neutral polyamide linkages. The 2-aminoethyl-lycine polyamide
linkage in particular has been well-studied and shown to impart
exceptional hybridization specificity and affinity when nucleobases
are attached to the linkage through an amide bond (Buchardt, 1992;
Nielsen, 1991).
[0007] 2-Aminoethylglycine PNA oligomers (FIG. 1A) typically have
greater affinity, i.e. hybridization strength and duplex stability
for their complementary PNA, DNA and RNA, as exemplified by higher
thermal melting values (Tm), than the corresponding DNA sequences.
The melting temperatures of PNA/DNA and PNA/RNA hybrids are much
higher than corresponding DNA/DNA or DNA/RNA duplexes (generally
1.degree. C. per bp) due to a lack of electrostatic repulsion in
the PNA-containing duplexes. Also, unlike DNA/DNA duplexes, the Tm
of PNA/DNA duplexes are largely independent of salt concentration.
The 2-aminoethylglycine PNA oligomers also demonstrate a high
degree of base-discrimination (specificity) in pairing with their
complementary strand. Specificity of hybridization can be measured
by comparing Tm values of duplexes having perfect Watson/Crick
complementarity and those with one or more mismatches. The degree
of destabilization of mismatches, measured by the decrease in Tm
(.DELTA.Tm), is a measure of specificity. In addition to
mismatches, specificity and affinity are affected by structural
modifications, hybridization conditions, and other experimental
parameters. The neutral backbone of PNA also increases the rate of
hybridization significantly in assays where either the target,
template, or the PNA probe is immobilized on a solid substrate.
Without any electrostatic repulsion, the rate of hybridization is
often much higher for PNA probes than for DNA or RNA probes in
applications such as Southern blotting, northern blots, or in situ
hybridization experiments (Corey, 1995). Unlike DNA, PNA can
displace one strand. "strand invasion", of a DNA/DNA duplex (Kuhn,
1999). With certain DNA sequences, a second PNA can further bind to
form an unusually stable triple helix structure (PNA).sub.2/DNA.
PNA have been investigated as potential antisense agents, based on
their sequence-specific inhibition of transcription and translation
(Von Matt, 1999; Lee, 1998, Nielsen, 1996). PNA oligomers
themselves are not substrates for polymerase as primers or
templates, and do not conduct primer extension with nucleotides
(Demers, 1997, see col. 2, lines 55-56).
[0008] PNA-DNA chimera are oligomer molecules with discrete PNA and
nucleotide moieties. They can be synthesized by covalently linking
PNA monomers and nucleotides in virtually any combination or
sequence. Efficient and automated methods have been developed for
synthesizing PNA-DNA chimera (Vinayak, 1997: Uhlmann, 1996: Van der
Laan, 1997). PNA-DNA chimera are designed to have desirable
properties found in PNA and DNA, e.g. superior hybridization
properties of PNA and biological functions like DNA (Uhlmann,
1998).
[0009] Attempts to demonstrate primer extension of PNA-DNA chimeric
primers with radioisotopically-labelled nucleotides have been
reported. Primer extension on an 81 nt DNA template was attempted
from a complementary PNA-DNA chimera with 15 PNA monomer units
linked through an amide bond to a single 3' terminal thymidine
nucleoside (FIG. 1B), various polymerases, and nucleotides dATP,
dGTP, dTTP, and .sup.32P-dCTP (Lutz, 1998). Some incorporation of
nucleotides and extension may be evident, but due to the
unavailability of proper control experiments, the level of
incorporation is unknown.
[0010] Primer extension was also reported using a mixture of
PNA-DNA chimera consisting of 19 PNA monomer units with three (FIG.
1D) and four 2'-deoxynucleotides, labelled once and twice
respectively, with .sup.32P dCTP and terminal transferase (Misra,
1998). The 3' hydroxyl terminus was extended on a 49 nt DNA
template and a 30 nt RNA template with unlabelled nucleotide
5'-triphosphates. Autoradiography of the gel after electrophoresis
showed a ladder of radiolabelled products, the majority of which
was unextended chimera, indicating inefficient primer extension.
This experiment employed a relatively long PNA moiety, 19 monomer
units, incurring the attendant costs, loss of specificity, and
synthesis inefficiencies of a longer chimera oligomer.
[0011] In another study, chimera consisting of 3 PNA monomer units
and either 2,4,6,9, or 12 deoxynucleotides were extended with
Klenow polymerase from an 18 nt DNA template (Reeve, 1995). All
chimera had T deoxynucleotide at the linkage between the PNA and
DNA moieties. Detection of incorporated .sup.32P-dCTP by
autoradiography indicated that all the chimera except the one with
2 deoxynucleotides were extended. However, no quantitative or
qualitative data was provided. Given the sensitivity of
autoradiography, extension of the chimera in this study may have
been at a detectable, but not useful, level.
[0012] Fluorescence has largely supplanted radioactivity as the
preferred detection method for most primer extension applications,
such as automated DNA sequencing, in vitro DNA probe-based
diagnostics, nucleic acid amplification, DNA fragment analysis, and
transcriptional expression mapping and profiling. It is thus
desirable to provide methods by which PNA-DNA chimera can be
enzymatically extended to form non-radioisotopically labelled
extension products. DNA sequencing methods benefit from the use of
PNA-DNA chimera as primers, in particular where the template is
double-stranded or where random priming is conducted with an array
of a large number of chimera, or mixed-base sequence chimera. The
increased affinity and specificity conferred by the PNA moiety in a
PNA-DNA chimera allows for shorter primers. Shorter primers are
more economical and require less sequence information. Such methods
would improve assays and tests based on primer extension, e.g.
greater precision and accuracy.
III. SUMMARY
[0013] The invention relates to chimera molecules of PNA and DNA
monomer units and their use in primer-extension methods, such as
DNA sequencing and nucleic acid amplification, to generate
non-radioisotopically labelled extension products.
[0014] The invention provides methods for enzymatic extension of
PNA-DNA chimera primers to generate labelled primer extension
products. The invention is based on the discovery that a PNA-DNA
chimera can conduct primer extension under a broad range of
experimental conditions and variables. PNA-DNA chimeras of the
invention include two moieties covalently linked together: i) a
contiguous moiety of 5 to 15 PNA monomer units, and ii) a
contiguous moiety of at least three nucleotides. The nucleotide
moiety has an enzymatically-extendable terminus, such that the
PNA-DNA chimera can be enzymatically extended.
[0015] In a first aspect, the invention provides a method of
producing a template-dependent, non-radioisotopically labelled
chimeric extension product by enzymatically extending a PNA-DNA
chimera primer annealed to a template nucleic acid in the presence
of a polymerase and a primer extension reagent (FIG. 2). The primer
extension reagent comprises a nucleotide 5'-triphosphate capable of
supporting template-dependent extension. The chimera and/or the
nucleotide 5'-triphosphate may be labelled with a non-radioisotopic
label such that the extension products are non-radioisotopically
labelled. In one illustrative embodiment of the invention, the
PNA-DNA chimera has the formula: P.sub.x-L-N.sub.y3', where each P
is independently a PNA monomer, x is an integer from 5 to 15, L
represents a covalent linkage between P and N, each NT is
independently a nucleotide y is an integer from 3 to 15. and the 3'
terminal N has a 3' hydroxyl group (FIG. 1D).
[0016] In one embodiment of the method, the extension reagent
comprises a mixture of nucleotide 5'-triphosphates capable of
incorporation and creation of an extended primer with a 3'
hydroxyl, and capable of further, continuous extension, e.g.
2'-deoxyribonucleotides (dNTP) and ribonucleotides (NTP). The
reagent may further include one or more terminator nucleotides
capable of incorporation, e.g. 2',3'-dideoxynucleotides (ddNTP) and
2,3'-dideoxy-dehydronucleotides, that, once incorporated, terminate
further extension. In another embodiment, the reagent comprises
only terminator nucleotides and does not include nucleotide
5'-triphosphates capable of continuous extension.
[0017] In a preferred embodiment, the PNA moiety, i.e., P.sub.x, of
the PNA-DNA chimera is a 2-aminoethylglycine peptide nucleic
acid.
[0018] The DNA moiety, i.e., N.sub.y, of the PNA-DNA chimera may be
comprised of 2'-deoxynucleotides (DNA), ribonucleotides (RNA), and
modified sugars or internucleotide linkages thereof, especially
those that confer greater specificity, affinity, rates of
hybridization, and chemical stability.
[0019] In embodiments employing a labelled PNA-DNA chimera, the
PNA-DNA chimera may be labelled at: (i) a nucleobase, e.g. the N-9
or C-8 positions of a purine or a deazapurine nucleobase, or the
C-5 position of a pyrimidine nucleobase; (ii) a sugar; (iii) the
aminoethylglycine backbone; or (iv) an amino, a sulfide, a
hydroxyl, and/or a carboxyl group. Preferably, the chimera is
labelled at the amino terminus of the PNA moiety. In embodiments
employing a labelled nucleotide, the nucleotide 5'-triphosphate is
preferably labelled at the nucleobase, but may also be labelled at
other positions provided that the label does not interfere with
enzymatic incorporation. Labels may be fluorescent dyes (FIGS.
3A-3B), fluorescence quenchers (FIG. 4), hybridization-stabilizers-
, energy-transfer dye pairs, electrophoretic mobility modifiers,
chemiluminescent dyes, amino acids, proteins, peptides, enzymes,
and affinity ligands. Preferably, the label is detectable upon
illumination with light, e.g. laser sources at infrared, visible or
ultraviolet excitation wavelengths.
[0020] The linkage, L, between the PNA and DNA moieties is
preferably a bond, e.g. the carbonyl-nitrogen bond in an amide
group where the moieties are linked without intervening atoms
(FIGS. 1B-1D). In another embodiment, the linkage may be a
multi-atom linker, e.g. an alkyldiyl consisting of 1 to 20 carbon
atoms, an aryldiyl consisting of 6 to 20 carbon atoms, optionally
including one or more heteroatoms, 0 linker, or 1 to 6 ethyleneoxy
units, --(CH.sub.2CH.sub.2O)-- (FIGS. 6A-6B).
[0021] The template or target nucleic acid can be any nucleic acid
or nucleic acid analog capable of mediating template-directed
nucleic acid synthesis. Examples of suitable template nucleic acids
include, e., genomic DNA, DNA digests, plasmids, vectors, viral
DNA, PCR products, RNA, and synthetic nucleic acids. The template
nucleic acid may also be a metaphase or interphase chromosome.
Preferably, the chromosome is denatured prior to PNA-DNA chimera
hybridization and primer extension. Template nucleic acids may be
single-stranded or double-stranded. Templates are typically larger
than the PNA-DNA chimera primer and can range from as few as about
20-30 to as many as millions of nucleotides (nt) or base-pairs
(bp), depending on the particular application.
[0022] Suitable enzymes to extend the PNA-DNA chimera primers
depend on the composition of the template nucleic acid. Reverse
transcriptases may be used for extending RNA templates, e.g. mRNA.
DNA polymerase may be used for extending DNA templates.
[0023] The template nucleic acid or the PNA-DNA chimera may be
immobilized on a solid substrate. When immobilized, the template or
chimera is preferably covalently attached to the solid substrate,
e.g. via a terminal monomer unit.
[0024] In a second aspect of the invention, a kit for primer
extension is provided which comprises: (i) a PNA-DNA chimera having
from 5 to 15 contiguous PNA monomer units, from 3 to 15 continuous
nucleotides, and a 3' hydroxyl terminus; (ii) one or more
enzymatically extendable nucleotide 5'-triphosphates and, (iii) a
polymerase enzyme. The chimera and/or a nucleotide 5'-triphosphate
is labelled with a non-radioisotopic label. In another embodiment,
the kit additionally includes a template comprising a sequence
complementary to the chimera or containing one or more mismatches
to the chimera.
[0025] In a third aspect, the invention provides methods for
sequencing a template nucleic acid by enzymatically extending a
PNA-DNA chimera primer hybridized to the template in the presence
of a polymerase and a terminating nucleotide 5'-triphosphate. The
chimera or the nucleotide 5'-triphosphate is non-radioisotopically
labelled. Generally, the methods for sequencing a template nucleic
acid comprise the steps of. (i) generating a series of
differently-sized primer extension products by enzymatically
extending a PNA-DNA chimera annealed to the template nucleic acid
in the presence of a polymerase, a mixture of nucleotide
5'-triphosphates capable of supporting continuous primer extension
and at least one terminating nucleotide 5'-triphosphate; (ii)
separating the primer extension product from one another, typically
based on size; and (iii) determining the sequence of the template
nucleic acid.
[0026] In one embodiment, a nested set of labelled primer extension
products, i.e. a set in which each extension product is one nt
shorter than the preceding extension product of the set, are
generated by a mixture of enzymatically-extendable nucleotide
5'-triphosphates capable of supporting continuous primer extension.
Where either the PNA-DNA chimera or the terminating nucleotide
5'-triphosphate bears a non-radioisotopic label, the identity of
certain bases is revealed by the labels, as is well known in the
art of nucleic acid sequencing (Bergot, 1994; Lee, 1992, Smith,
1998). In one convenient embodiment, a mixture of four different
labelled terminating nucleotides is used, e.g. ddATP, ddCTP, ddGTP,
ddTTP, each bearing a different, determinable label, such that the
3'-terminus nucleotide of each primer extension product is revealed
by the identity of the label. Alternatively, the method can be
performed in the absence of nucleotide 5'-triphosphates capable of
supporting continuous primer extension such that only a single
terminating nucleotide is added (Goelet, 1999; Syvanen, 1990).
[0027] The methods of the present invention are well-suited to
fluorescent detection, particularly the simultaneous detection of
multiple spectrally-resolvable fluorescent dyes. The methods are
particularly well-suited for detecting, identifying, or determining
classes of primer extension products that have been subjected to a
separation procedure, such as electrophoresis, or that have been
distributed amongst locations in a spatially-addressable
hybridization array.
[0028] In a fourth aspect, a method is provided for reverse
transcription of RNA by enzymatically extending a primer-template
RNA hybrid in the presence of a reverse transcriptase and a mixture
of enzymatically-extendable nucleotide 5'-triphosphates capable of
supporting continuous primer extension. The primer is a PNA-DNA
chimera and either said primer or a nucleotide 5'-triphosphate is
non-radioisotopically labelled.
[0029] In a fifth aspect, a method is provided for in situ
chromosome targetting by single or multiple labelling where the
chromosome is denatured and either in metaphase or interphase. A
primer-chromosome hybrid is enzymatically extended in the presence
of a DNA polymerase and a mixture of nucleotide 5'-triphosphates
capable of supporting continuous primer extension. The primer is a
PNA-DNA chimera. Either the primer or a nucleotide 5'-triphosphate
is non-radioisotopically labelled. Fluorescence at the chimera
binding sites on the chromosome can be detected.
[0030] In a sixth aspect, a method is provided for DNA
amplification where amplification products are generated by
enzymatically extending a primer-template nucleic acid in the
presence of two primers, a DNA polymerase and a mixture of
enzymatically-extendable nucleotide 5'-triphosphates capable of
supporting continuous primer extension. One or both of the primers
is a PNA-DNA chimera. Either the primers or nucleotide
5'-triphosphates are non-radioisotopically labelled. The
temperature is cycled to effect denaturation, annealing, and primer
extension to form an amplification product by extension of the
primers with the nucleotide 5'-triphosphates. One or both of the 5'
terminii of the amplification product bears the PNA sequence of the
chimera primers.
[0031] In one embodiment, the amplification product is immobilized
by hybridization on a solid substrate comprising a nucleic acid
having a sequence complementary to the PNA moiety of the
amplification product.
[0032] These and other aspects, objects, features, and advantages
of the present invention will become better understood with
reference to the following description, drawings, examples, and
appended claims.
IV. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 Structures of N-(2-aminoethyl)-glycine PNA (IA) and
PNA-DNA chimera with one (1B), two (1C), and three (1D)
2'-deoxynucleotides, where B is a nucleobase. The wavy line segment
represents continuing, repeating units.
[0034] FIG. 2 Schematic of extension of PNA-DNA chimera on a
template nucleic acid.
[0035] FIG. 3 Fluorescent dye label structures: FAM, TET, HEX, JOE,
NED, VIC (3A); dJON, dR139, JODA, FAM donor (3B). X denotes an
attachment site.
[0036] FIG. 4 Quencher label structures: TAMRA, ROX, DABCYL,
DABSYL, NTB. X denotes an attachment site. X denotes an attachment
site. Z is H or NO.sub.2.
[0037] FIG. 5 Linker reagents for linkers between PNA and DNA
moieties in chimera.
[0038] FIG. 6 PNA-DNA chimera with bis-ethyleneoxy-acetamido linker
(6A) and bis-ethyleneoxy-phosphate linker (6B).
[0039] FIG. 7 Polyacrylamide (15%) gel electrophoresis under
denaturing conditions (7M urea) and SYBR-Green staining. Primer
extension of a DNA 38 nt template (SEQ. ID NO. 8) with various
primers. Top panel: Klenow (exo-) polymerase, middle panel:
AmpliTaq FS polymerase, bottom panel: no enzyme
1 Lane Primer (SEQ. ID NO.) 1 none 2 TAG TTC 1 3 TAG TTC-t 2 4 TAG
TTC-ta 3 5 TAG TTC-tag 4 6 TAG TTC-taga 5 7 5' tag ttc 3' 6 8 5'
tag ttc tag 3' 7 M DNA oligo ladder
[0040] PNA--UPPER CASE; DNA--lower case
[0041] FIG. 8 Polacrylamide (15%) gel electrophoresis under
denaturing conditions (7M urea) and SYBR-Green staining. Primer
extension of DNA 38 nt template (SEQ ID NO. 8) with PNA-DNA chimera
with Klenow (exo-) polymerase
2 Lane Primer (SEQ. ID NO.) 1 Ac-TAG TTC T - ag 9 2 Ac-TAG TTC T -
aga 10 3 Ac-TAG TTC T - agac 11
[0042] FIG. 9 Polyacrylamide (15%) gel electrophoresis under
denaturing conditions (7M urea) and SYBR-Green staining.
Specificity of DNA and PNA-DNA chimera primers with Klenow (top
gel) and Bst polymerase (bottom gel), and perfect match 38 nt
template (SEQ. ID NO. 8) (left side) and two mismatched templates
(SEQ. ID NO. 12 and 13) (right side).
3 Lane Primer (SEQ. ID NO.) 6/0 TAG TTC 1 6/1 TAG TTC - t 2 6/2 TAG
TTC - ta 3 6/3 TAG TTC - tag 4 6/4 (6/4') TAG TTC - taga 5 0/6 5'
tag ttc 3' 6 0/9 5' tag ttc tag 3' 7 DNA oligo ladder
[0043] FIG. 10 Primer sequences on left side panels of FIG. 9 in
perfect match priming of 38 nt DNA template (SEQ. ID NO. 8). (upper
case--PNA, lower case--DNA)
[0044] FIG. 11 Primer sequences on right side panels of FIG. 9 in
mismatched priming of: 38 nt DNA template (SEQ. ID NO. 12) with
one-base mismatch at the 2nd base (c), complementary to the 2nd
base after the PNA-DNA linkage; and 38 nt DNA template (SEQ. ID NO.
13) with one-base mismatch at the 4th base (c), complementary to
the 4th base after the PNA-DNA linkage in chimera primer 6/4'.
(upper case--PNA, lower case--DNA)
[0045] FIG. 12 MALDI-TOF mass spectroscopy of primer extension
product, sample from lane 6, FIG. 7. MW 8750.7 of fully extended
PNA.sub.6DNA.sub.23 product.
[0046] FIG. 13 Polyacrylamide gel electrophoresis under denaturing
conditions (7M urea) and SYBR-Green staining. Mouse Xist gene mRNA
was reverse transcribed with PNA-DNA chimera primer. The cDNA copy
was amplified with D2 and D3 primers to give 143 bp and 210 bp
fragments.
[0047] FIG. 14 Schematic of single nucleotide polymorphism (SNP)
detection by one-base PNA-DNA chimera primer extension from wild
type (WT) and mutant templates, and MALDI-TOF mass spectral
analysis of extension products.
[0048] FIG. 15 Polyacrylamide (15%) gel electrophoresis under
denaturing conditions with fluorescence detection (no staining).
Detection of fluorescent labelled primer extension products by
incorporation of TAMRA-dUTP with PNA-DNA chimera primer (SEQ. ID
NO. 4) and 5' biotin DNA 38 nt template (SEQ. ID NO. 8).
V. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0049] Reference will now be made in detail to the preferred
embodiments of the invention. While the invention will be described
in conjunction with the preferred embodiments, it will be
understood that they are not intended to limit the invention to
those embodiments. On the contrary, the invention is intended to
cover alternatives, modifications, and equivalents, which may be
included within the invention as defined by the appended
claims.
[0050] Generally, the present invention comprises primer extension
methods where the primer is a PNA-DNA chimera. The methods of the
present invention find particular application in the area of
nucleic acid analysis, e.g. DNA sequencing, fragment analysis, and
detection of probe hybridization in hybridization assays.
V. 1 Definitions
[0051] Unless stated otherwise, the following terms and phrases as
used herein are intended to have the following meanings:
[0052] "Nucleobase" refers to a nitrogen-containing heterocyclic
moiety, e.g. a purine, a 7-deazapurine, or a pyrimidine. Typical
nucleobases are adenine, guanine, cytosine, uracil, thymine,
7-deazaadenine, 7-deazaguanine, and the like.
[0053] "Nucleoside" refers to a compound consisting of a nucleobase
linked to the C-1' carbon of a ribose sugar.
[0054] "Nucleotide" refers to a phosphate ester of a nucleoside, as
a monomer unit or within a nucleic acid. Nucleotides are sometimes
denoted as "NTP", or "dNTP" and "ddNTP" to particularly point out
the structural features of the ribose sugar. "Nucleotide
5'-triphosphate" refers to a nucleotide with a triphosphate ester
group at the 5' position. The triphosphate ester group includes
sulfur substitutions for the various oxygens, e.g.
.alpha.-thio-nucleotide 5'-triphosphates.
[0055] As used herein, the term "nucleic acid" encompasses the
terms "oligonucleotide" and "polynucleotide" and means
single-stranded and double-stranded polymers of nucleotide
monomers, including 2'-deoxyribonucleotides (DNA) and
ribonucleotides (RNA). The nucleic acid may be composed entirely of
deoxyribonucleotides, entirely of ribonucleotides, or chimeric
mixtures thereof. The monomers are linked by internucleotide
phosphodiester bond linkages, and associated counterions, e.g.,
H.sup.+, NH.sub.4.sup.+, trialkylammonium, Mg.sup.2+, and Na.sup.+.
Nucleic acids typically range in size from a few monomeric units,
e.g. 5-40 when they are commonly referred to as oligonucleotides,
to several thousands of monomeric units. Whenever an
oligonucleotide sequence is represented, it will be understood that
the nucleotides are in 5' to 3' order from left to right and that
"A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes
deoxyguanosine, and "T" denotes thymidine, unless otherwise
noted.
[0056] The term "Watson/Crick base-pairing" refers to the
hydrogen-bonding base pairing commonly observed in double-stranded
DNA.
[0057] "Attachment site" refers to a site on a moiety, e.g. a
chimera or nucleotide, to which is covalently attached a
linker.
[0058] "Linker" refers to one or more atoms used to space one
moiety from another, e.g. a label from a nucleotide 5'-triphosphate
or the PNA moiety from a DNA moiety in a PNA-DNA chimera.
[0059] "PNA-DNA Chimera" refers to an oligomer, or oligomers,
comprised of: (i) a contiguous moiety of PNA monomer units and (ii)
a contiguous moiety of nucleotide monomer units with an
enzymatically-extendable terminus.
[0060] "Alkyl" refers to a saturated or unsaturated, branched,
straight-chain, branched, or cyclic hydrocarbon radical derived by
the removal of one hydrogen atom from a single carbon atom of a
parent alkane, alkene, or alkyne. Typical alkyl groups include, but
are not limited to, methyl, ethyl, propyl, butyl, and the like. In
preferred embodiments, the alkyl groups consist of 1-12 saturated
and/or unsaturated carbons.
[0061] "Cycloalkyl" refers to a cyclic alkyl radical. Nitrogen
atoms with cycloalkyl substituents may form aziridinyl, azetidinyl,
pyrrolidinyl, piperidinyl, larger rings, and substituted forms of
heterocycles thereof.
[0062] "Alkyldiyl" refers to a saturated or unsaturated, branched,
straight chain or cyclic hydrocarbon radical of 1-20 carbon atoms,
and having two monovalent radical centers derived by the removal of
one hydrogen atom from each of two different carbon atoms of a
parent alkane, alkene or alkyne. Typical alkyldiyl radicals
include, but are not limited to, 1,2-ethyldiyl, 1,3-propyldiyl,
1,4-butyldiyl, and the like.
[0063] "Aryldiyl" refers to an unsaturated cyclic or polycyclic
hydrocarbon radical having a conjugated resonance electron system
and at least two monovalent radical centers derived by the removal
of one hydrogen atom from each of two different carbon atoms of a
parent aryl compound. Typical aryldiyl groups include, but are not
limited to, radicals derived from benzene, substituted benzene,
naphthalene, anthracene, biphenyl, and the like.
[0064] "Label" refers to any non-radioisotopic moiety covalently
attached to a chimera or nucleotide that is detectable or imparts a
desired functionality or property in the primer extension product
(Hermanson, 1996). Preferred detectable labels are fluorescent
dyes.
[0065] "Primer extension" is the enzymatic addition, i.e.
polymerization, of monomeric nucleotide units to a primer while the
primer is hybridized (annealed) to a template nucleic acid.
V.2 PNA-DNA Chimera
[0066] PNA-DNA chimera are linear oligomers comprised of: 1) a
contiguous moiety of PNA monomer units and 2) a continuous moiety
or nucleotides. The two moieties are covalently linked together.
The nucleotide moiety of the chimera may be 2'-deoxynucleotides,
ribonucleotides, or a mixture thereof. The nucleotide moiety of the
chimera has a 3' hydroxyl terminus. The preferred length of the PNA
moiety is from 5 to 15 PNA monomer units, reflecting optimum
enzymatic activity, hybridization specificity and affinity, economy
of synthesis reagents, and ease of chimera synthesis and
purification. The length of the DNA moiety is from 3 to 15
nucleotides. The preferred length of the DNA moiety is the shortest
sequence which promotes efficient primer extension, i.e. at least
three 2'-deoxynucleotides (FIG. 1D).
[0067] A preferred form of the PNA moiety is an uncharged backbone
of N-(2-aminoethyl)-glycine, a peptide-like, amide-linked unit
(Egholm, 1993; Nielsen, 1991) (FIG. 1A). Whenever a PNA sequence is
represented as a series of letters, it is understood that the amino
terminus is at the left side and the carboxyl terminus is at the
right side.
[0068] Binding of the PNA moiety to its DNA or RNA complement can
occur in either a parallel or anti-parallel orientation of PNA;
however, the anti-parallel duplex (where the carboxyl terminus of
PNA is aligned with the 5' terminus of DNA, and the amino terminus
of PNA is aligned with the 3' terminus of DNA) is typically more
stable (Egholm, 1993). The chimera of the present invention are
designed such that the PNA moiety anneals in the anti-parallel
orientation with the target sequences.
[0069] Chimera sequences are typically completely complementary to
a portion of the target sequence. However, chimera sequences may
contain mixed-base ("redundant" or "degenerate") sites whereby a
chimera sample may be a mixture of sequences with one or more sites
represented by two or more different nucleobases. The mixed-base
site may be located in the PNA or DNA moieties of the oligomer.
Mixed-base chimera are mixtures of sequences with varying levels of
complementarity to a particular target sequence. Mixed-base chimera
may be useful for random priming or where template sequence
information is unknown or uncertain.
[0070] PNA-DNA chimera can be synthesized using the respective
conventional methods of synthesis of PNA oligomers, DNA
oligonucleotides, and RNA oligonucleotides. Chimera can be
synthesized at a 2-25 .mu.mole scale on commercially available,
automated synthesizers, e.g. Expedite, Model 433A and Model 394
Synthesizers (PE Biosystems), and with commercially available
reagents (Uhlmann, 1996; Vinayak, 1997; Van der Laan, 1997). In
this approach, the chimera can be made continuously, in a single
column and on a single synthesizer.
[0071] Synthesis of chimera is initiated by detritylation of the 5'
dimethoxytrityl (DMT) group of commercially available, high-cross
link, non-swelling polystyrene beads packed in a synthesis column.
The supports are loaded at 20-30 .mu.mole/gm with 5' DMT
deoxynucleosides (A.sup.bz, G.sup.ibu, C.sup.bz, T) linked through
the 3 hydroxyl to the support through a base-labile
succinate/hydroxymethylbenzoic acid linker (Vinayak, 1997). 5' DMT,
3' cyanoethyl phosphoramidite deoxynucleoside monomers (Beaucage,
1992) are dissolved in dry acetonitrile and delivered concurrently
with tetrazole activator and coupled to the support-bound 5'
hydroxyl. Coupling is followed by capping with acetic anhydride of
unreacted 5' hydroxyls, and iodine oxidation to the pentavalent
internucleotide phosphate triester. The DNA synthesis cycle is
repeated until the last deoxynucleoside addition, where a 5'
monomethoxytrityl (MMT) amino nucleoside phosphoramidite is
employed to furnish a 5' amino terminus on the support-bound DNA
moiety, for coupling to a PNA monomer at the linkage between DNA
and PNA in the chimera. The MMT group is favored in the synthesis
of PNA-DNA chimera because of its acid-lability. The MMT group is
efficiently and rapidly removed under mild acidic conditions which
do not cause depurination or other damage to the chimera.
[0072] To initiate synthesis of the PNA moiety, the 5' MMT group is
removed with 3% trichloroacetic acid in dichloromethane and the
amino group is coupled with a PNA monomer and a coupling reagent.
The backbone amino group of the PNA monomers is preferably
protected with MMT and the nucleobase exocyclic amines are
protected as A.sup.bz, G.sup.ibu, and C.sup.bz (Breipohl, 1997;
Finn, 1996; Will, 1995) Any conventional peptide coupling reagent
may be used, but HBTU and HATU are preferred coupling reagents. PNA
monomers may be dissolved in 1:1 DMF:acetonitrile to a
concentration of about 0.2M. Prior to delivery to the synthesis
column, the monomer solution was mixed with an equal volume of 0.2M
HBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
hexafluorophosphate), also in 1:1 DMF:acetonitrile (Vinayak. 1997).
The solution was delivered to the column concurrently with 0.2M
diisopropylethylamine in 1:1 DMF:acetonitrile. The synthesis cycles
for the PNA and DNA moieties in a chimera are summarized in Table 1
below.
4TABLE 1 Synthesis cycles for PNA and DNA moieties of PNA-DNA
chimera. PNA DNA Step Function Reagents Time (sec) Time (sec) 1
Detritylation 3% CCl.sub.3CO.sub.2H in 60 25 CH.sub.2Cl.sub.2 2
Coupling PNA: 0.2 M PNA 960 25 monomer, HBTU, DiPEAin 1:1
DMF:CH.sub.3CN DNA: 0.1 M DNA monomer, 0.5 M tetrazole in
CH.sub.3CN 3 Capping Ac.sub.2O, lutidine, N- 25 15 methylimidazole,
THF 4 Oxidation iodine, pyridine, not required 25 H.sub.2O, THF
[0073] Model 394 synthesizer, 2 .mu.mole scale.
[0074] After synthesis is complete, the amino terminus may be
acetylated to minimize migration or cyclization, or reacted as a
nucleophile in labelling. The crude chimera is cleaved from the
support, and all protecting groups are removed with concentrated
ammonium hydroxide at 55.degree. C. for 8-16 hours. The chimera are
analyzed and purified by reverse-phase HPLC or polyacrylamide gel
electrophoresis (PAGE), analyzed by mass spectroscopy, and
quantitated by correlating UV absorbance at 260 nm with mass.
[0075] Chimera with a DNA moiety comprising ribonucleotides can be
synthesized with the appropriate RNA phosphoramidite nucleosides
and/or 5' DMT protected ribonucleotides support (Vinayak, 1994).
The 2' hydroxyl of RNA phosphoramidites are typically protected
with the tert-butyldimethylsilyl (TBDMS) group and the exocyclic
amino groups of the nucleobases are protected as A.sup.bz,
G.sup.dmf, C.sup.bz. After synthesis, TBDMS groups are removed with
a fluoride reagent, e.g. tetrabutylammonium fluoride in
tetrahydrofuran. Otherwise, the synthesis, purification, and
analysis methods for ribonucleotide-containing PNA-DNA chimera are
virtually the same as for chimera with only 2'-deoxynucleotide
containing DNA moieties.
[0076] The PNA and DNA moieties are covalently linked together. The
linkage may be a direct bond, e.g. an amide bond formed by the
amino group at the 5' of a deoxynucleotide and the carboxyl group
at the carboxyl terminal of the PNA moiety. The linkage may also
comprise one or more units of a non-base pairing moiety such
ethyleneoxy, linked to the PNA and DNA moieties by amide or
phosphate bonds. Ethyleneoxy linkage units between the PNA and DNA
moieties can be installed by coupling reagents such as protected
forms of O-[2-(2-aminoethoxy)ethoxy]acetic acid. The 0-linker,
2-[2-aminoethoxy]acetic acid, is coupled as the MMT-amino protected
amide-forming carboxylic acid, or phosphoramidite synthons (FIG.
5). One or more O linker units act as a flexible, non-base pairing,
linkage between the PNA and DNA moieties. FIG. 6 shows a
bis-ethyleneoxy-acetamido linker (6A) and a
bis-ethyleneoxy-phosphate linker (6B). Other linkers include
alkydiyl, e.g. hexyldiyl (Vinayak, 1997), or 1,4-phenyldiyl (FIG.
5).
V.3 Nucleotide 5'-Triphosphates
[0077] Nucleotide 5'-triphosphates are substrates of polymerase
enzymes and are incorporated into the template/chimera hybrid by
internucleotide phosphodiester bond formation between the 3'
hydroxyl terminus of the chimera and the 5' hydroxyl of the
nucleotide. Further extension by incorporation of more nucleotide
5'-triphosphates requires a new 3' hydroxyl terminus. During primer
extension, typically a mixture of nucleotide 5'-triphosphates are
present, e.g. dATP, dGTP, dCTP and dTTP. Labelled nucleotides may
also be present, for detection, isolation, or immobilization of the
extension fragments. Nucleotides which terminate extendability
("terminators" or "terminating nucleotides") may also be present in
the mixture, e.g. ddNTP and 2',3'-dehydro-ddNTP. Labelled
terminators are particularly useful. Individual concentrations of
each nucleotide in the mixture are optimized to promote the desired
incorporation rates and achieve the necessary detection levels.
Preferred nucleotide 5'-triphosphates of the present invention are
shown below in the general structures: 1
[0078] where B is a nucleobase. The 2' position of the ribose sugar
moiety may be substituted with 2'-O-alkyl, e.g. methyl, 2'-amino or
2'-halo. e.g. fluoro, chloro, as in the structure:. 2
[0079] where X is alkoxy, halo, and amino. When B is a purine or a
7-deazapurine, the sugar moiety is attached at the N.sup.9-position
of the purine or deazapurine, and when B is a pyrimidine, the sugar
moiety is attached at the N'-position of the pyrimidine.
[0080] Preferably the nucleotide 5'-triphosphate is ATP, dATP,
ddATP, CTP, dCTP, ddCTP, GTP, dGTP, ddGTP, UTP, dUTP, TTP, dTTP,
ddTTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, dITP, ddITP, 2-amino-ATP,
2-amino-dATP, 7-deaza dATP, 7-deaza ddATP, 5-propynyl dCTP, 7-deaza
dGTP, 7-deaza ddGTP, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP,
5-propynyl-dUTP. Additionally, the .alpha.-phosphorus may be
substituted with sulfur, as the .alpha.-thio-nucleotide
5'-triphosphates (Lee, 1992).
V.4 Polymerase Enzymes
[0081] A variety of polymerases, e.g. Vent (Kong, 1993), Klenow,
Bst, bacteriophage T7 DNA polymerase (Tabor, 1989) and its
processivity-enhancing protein partner, E. coli thioredoxin,
bacteriophage T4 DNA polymerase and its processivity clamp, gp45
protein (Carver, 1997), Taq, and Sequenase conduct primer extension
of PNA-DNA chimera. Preferred polymerases include Vent, Klenow and
Bst. Polymerases without exo activity (Exo.sup.-) proof reading
function are preferred.
[0082] Reverse transcriptase enzymes extend PNA-DNA chimera from
RNA templates to make cDNA copies with nucleotide 5'-triphosphates.
Preferred reverse transcriptases are from avian myeloblastosis
virus (AMV) and murine leukemia virus (MuLV) and HIV.
V.5 Nucleotides
[0083] Preferred nucleobases in one or more nucleosides include,
but are not limited to, adenine, guanine, cytosine, uracil,
thymine, 7-deazaadenine. 7-deazaguanine, C-5-alkyl pyrimidines,
2-thiopyrimidine, 2.6-diaminopurine, C-5-propyne pyrimidine,
phenoxazine (Flanagan, 1999), 7-deazapurine, isocytidine,
pseudo-isocytidine (Egholm, 1995), isoguanosine, 4(3 H)-pyrimidone,
hypoxanthine, and 8-oxopurines (Meyer, 1994).
[0084] Preferred sugars in one or more of the nucleosides include,
but are not limited to, 2'-deoxyribose, ribose, and 2'- or
3'-ribose modifications where the 2'- or 3'-position may be
hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy, butoxy,
isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino,
fluoro, chloro and bromo.
[0085] Other preferred sugars include 4'-.alpha.-anomeric
nucleotides, 1'-.alpha.-anomeric nucleotides, and 2'-4'-linked and
other "locked", bicyclic sugar modifications (Wengel, 1999).
V.6 Labels
[0086] The PNA-DNA chimera or the nucleotide 5'-triphosphates may
bear covalently attached non-radioisotopic labels. The chimera and
one or more of the nucleotide 5'-triphosphates in a primer
extension reaction may bear the same or different labels. Labeling
can be accomplished using any one of a large number of known
techniques employing known labels, linkages, linking groups,
reagents, reaction conditions, and analysis and purification
methods. Generally, the linkage linking the dye and nucleotide or
chimera should not (i) interfere with primer extension, (ii)
inhibit polymerase activity, or (iii) adversely affect the
fluorescence properties of the dye, e.g. quenching or
bleaching.
[0087] PNA-DNA chimera and nucleotide 5'-triphosphates can be
labelled at sites including a nucleobase, a sugar, the
aminoethylglycine backbone, amino, sulfide, hydroxyl, and carboxyl.
Nucleobase label sites include the N-9 or C-8 positions of the
purine or deazapurine, and the C-5 position of the pyrimidine.
Preferably, the linkage between the label and the chimera or
nucleotide 5'-triphosphate are acetylenic amido or alkenic amido
linkages (Khan, 1998). Linkers can also comprise alkyldiyl,
aryldiyl, or one or more ethyleneoxy units (Rajur, 1997).
Typically, a carboxyl group on the label is activated by forming an
active ester, e.g. N-hydroxysuccinimide (NHS) ester and reacted
with an amino group on the alkynylamino- or
alkenylamino-derivatized chimera or nucleotide.
[0088] Labelled 2',3'-dideoxynucleotides, ddNTP, find particular
application as chain terminating agents, or "terminators" in the
Sanger-type DNA sequencing method of primer extension, and for
sizing/identification and analysis. Labelled deoxynucleotides,
dNTP, find particular application as means for labelling primer
extension products, e.g. in the polymerase chain reaction (Mullis,
1987).
[0089] A preferred class of labels provide a signal for detection
of labelled extension products by fluorescence, chemiluminescence,
and electrochemical luminescence (Kricka, 1992). Particularly
preferred chemiluminescent labels are 1,2-dioxetane compounds
(Bronstein, 1994; Bronstein, 1990). Fluorescent dyes useful for
labelling chimera and nucleotide 5'-triphosphates include
fluoresceins (Menchen, 1993), rhodamines (Bergot, 1994), cyanines
(Lee, 1998 (Ser. No. 09/012,525); Kubista, 1997), and metal
porphyrin complexes (Stanton, 1988).
[0090] Examples of fluorescein dyes include 6-carboxyfluorescein
(6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET),
2',4',5',7',1,4-hexachlorofluore- scein (HEX),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyrhodamine (JOE),
2'-chloro-5'-fluoro-7',8'-fused
phenyl-1,4-dichloro-6-carboxyfluorescein (NED),
2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and
(JODA) (FIGS. 3A-3B). The 5-carboxyl, and other regio-isomers, may
also have useful detection properties. Fluorescein and rhodamine
dyes with 1,4-dichloro substituents (bottom ring as shown) are
especially preferred.
[0091] Another preferred class of labels include fluorescence
quenchers. The emission spectra of a quencher overlaps with a
proximal intramolecular or intermolecular fluorescent dye such that
the fluorescence of the fluorescent dye is substantially
diminished, or quenched, by the phenomena of fluorescence resonance
energy transfer "FRET" (Clegg, 1992).
[0092] Particularly preferred quenchers include but are not limited
to (i) rhodamine fluorescent dyes selected from the group
consisting of tetramethyl-6-carboxyrhodamine (TAMRA),
tetrapropano-6-carboxyrhodamine (ROX), and (ii) DABSYL, DABCYL,
cyanine dyes including nitrothiazole blue (NTB), anthraquinone,
malachite green, nitrothiazole,and nitroimidazole compounds and the
like (FIG. 4). Nitro-substituted forms of quenchers are especially
preferred.
[0093] Energy-transfer dyes are a preferred class of
oligonucleotide labels. An energy-transfer dye label includes a
donor dye linked to an acceptor dye (Lee, 1998, U.S. Pat. No.
5,800,996), or an intramolecular FRET pair (Livak, 1998; Livak,
1996; Tyagi, 1996). Light, e.g. from a laser, at a first wavelength
is absorbed by a donor dye, e.g. FAM. The donor dye emits
excitation energy absorbed by the acceptor dye. The acceptor dye
fluoresces at a second wavelength, with an emission maximum
preferably about 100 nm greater than the absorbance maximum of the
donor dye.
[0094] The donor dye and acceptor dye of an energy-transfer label
may be directly attached by a linkage such as: 3
[0095] formed from an aminomethyl group at the 4' or 5' positions
of the donor dye, e.g. FAM and a 5- or 6-carboxyl group of the
acceptor dye (FIG. 3B). Other rigid and non-rigid linkers may be
useful.
[0096] Another preferred class of labels serve to effect the
separation or immobilization of labelled primer extension products
by specific or non-specific capture means, e.g. biotin,
2,4-dinitrophenyl (DNP), and digoxigenin (Andrus, 1995).
[0097] Another preferred class of labels are electrophoretic
mobility modifiers, e.g. polyethyleneoxy (PEO) units. The PEO label
may be comprised of charged groups, such as phosphodiester to
impart charge and increase electrophoretic mobility (velocity). The
PEO label may be uncharged and act to retard electrophoretic
mobility. Such modifiers may serve to influence or normalize the
electrophoretic velocity of a set of labelled primer extension
products during analysis, e.g. by fluorescent detection, to improve
resolution and separation (Grossman, 1995)
[0098] Another preferred class of labels, referred to herein as
hybridization-stabilizers, include but are not limited to minor
groove binders, intercalators, polycations, such as poly-lysine and
spermine, and cross-linking functional groups.
Hybridization-stabilizers may increase the stability of
base-pairing, i.e. affinity, or the rate of hybridization (Corey,
1995) of the chimera and the template. Hybridization-stabilizers
serve to increase the specificity of base-pairing, exemplified by
large differences in Tm between perfectly complementary
oligonucleotide and target sequences and where the resulting duplex
contains one or more mismatches of Watson/Crick base-pairing
(Blackburn, 1996, pp 15-81 and 337-46). Preferred minor groove
binders include Hoechst 33258 (Rajur, 1997 CDPI.sub.1-3 (Kutyavin,
1996), MGB1 (Gong, 1997),
V.7 Primer Extension
[0099] Primer extension is initiated at the template site where a
primer anneals. One or more different nucleotide 5'-triphosphates
may be present in the reaction mixture such the the complementary
nucleotide is incorporated by a polymerase enzyme according the
template sequence. Extension of the chimera continues until
nucleotides are depleted, the enzyme is no longer functional, or
termination occurs by incorporation of a terminating nucleotide
that will not support continued DNA elongation. Chain-terminating
nucleotides are typically 2',3'-dideoxynucleotide 5'-triphosphates
(ddNTP), which lack the 3'-OH group necessary for 3' to 5' DNA
chain elongation. Other terminating nucleotides include
2',3'-dideoxy-dehydro; 2'-acetyl; 2'-deoxy, halo; and other
2-substituted nucleotide 5'-triphosphates.
[0100] In general, the reaction conditions for primer extension
involve an appropriate buffering system to maintain a constant pH,
a divalent cation, a PNA-DNA chimera primer, a template nucleic
acid, nucleotide 5'-triphosphates, and a polymerase. Additional
primer extension reagents, such as reducing agents, monovalent
cations, or detergents may be added to enhance the reaction rate,
fidelity, or other parameters. Different polymerases may have
different optimal pH values or ion concentrations.
[0101] Klenow without exo activity (exo-) extended PNA-DNA chimera
primers comprised of six contiguous PNA monomers and three or four
contiguous 2'-deoxynucleotides (FIGS. 7 and 8). The same chimera
were not extended to give full length product by AmpliTaq FS.RTM.
polymerase (FIG. 7 middle panel). PNA-DNA chimera with
0-2,2'-deoxynucleotides were not extended by either enzyme (FIGS. 7
and 8). The identity of the full length 29 nt (PNA6DNA.sub.23)
extension product from lane 6 in FIG. 7 was confirmed by MALDI-TOF
mass spectroscopy (FIG. 12).
[0102] The specificity advantage of PNA-DNA chimera primers
relative to DNA primers is shown in FIG. 9. When various primers
were extended with Klenow (exo-) or Bst polymerases on a 38 nt DNA
template (SEQ. ID NO. 8) with perfect complementarity (FIG. 10),
chimera primers with three (6/3) and four (6/4) 2'-deoxynucleotides
were extended, as well as the corresponding all-DNA 9 nt primer
(0/9) The all-DNA hexamer (0/6) showed a weaker band under the
SYBR-Green staining detection. The all-DNA extension products from
0/6 and 0/9 primers migrated faster than the PNA-DNA extension
products from 6/3 and 6/4. However, when the template contained a
mismatch either across from the 2nd base (SEQ. ED NO. 12) or 4th
base (SEQ. ID NO. 13) from the PNA-DNA linkage site, the PNA-DNA
chimera did not extend (FIG. 11). The all-DNA 9 nt primer (0/9) did
extend, showing a band of near equal intensity to the perfect match
extension product. Thus while the all-DNA primer showed little
specificity, i.e. sequence discrimination of a mismatch, the
corresponding PNA-DNA chimera showed absolute specificity within
the detection limits of the experiment. The results of this
experiment follows other reports that PNA probes are more sensitive
to mismatches than DNA probes (Kyger, 1998).
[0103] Labeled primer extension products. "fragments", are
generated through template-directed enzymatic synthesis using
labeled chimera primers or nucleotides. The fragments can be
separated by a size-dependent process, e.g., electrophoresis or
chromatography; and the separated fragments detected, e.g., by
laser-induced fluorescence. In a preferred fragment analysis
method, Sanger-type sequencing, a chimera primer is extended by a
DNA polymerase in vitro using a single-stranded or double-stranded
DNA template whose sequence is to be determined. Extension is
initiated at a defined site based on where a chimera anneals to the
template. The extension reaction is terminated by incorporation of
a nucleotide that will not support continued DNA elongation, i.e. a
terminating nucleotide. When optimized concentrations of dNTP and
terminating nucleotides are used, enzyme-catalyzed polymerization
(extension) will be terminated in a fraction of the population of
chains at each site where the terminating nucleotide is
incorporated such that a nested set of primer extension fragments
result. If fluorescent dye-labeled chimera primers or labeled
terminating nucleotides are used for each reaction, the sequence
information can be detected by fluorescence after separation by
high-resolution electrophoresis (Smith, 1998). Each of the four
possible terminating nucleotides (A,G,C,T) may be present in the
extension reaction and bear a different fluorescent dye which are
spectrally resolvable (Bergot, 1994).
[0104] "Mini-sequencing" is another application involving
incorporation of terminating nucleotides in single-base extension
assays where PNA-DNA chimera may be useful to determine the
identity, presence, or absence of a nucleotide base at a specific
position in a nucleic acid target of interest (Goelet, 1999;
Syvanen, 1990). Genotype determination based on identification of
different alleles is based on single nucleotide polymorphisms
(SNP). SNP can be detected by ddNTP incorporation from PNA-DNA
chimera primers annealed immediately adjacent to the 3' of the SNP
site of the target nucleic acid sequence to be determined, and
detection of the extension products by MALDI-TOF mass spectroscopy
(FIG. 14). The mass difference resulting from incorporation of
different dideoxynucleotides can be accurately determined by mass
spectrometry. More than one chimera primer, each with a different
sequence and mass, allows detection of multiple SNP in a single
tube or reaction, by analyzing the mass spectra of the extension
products.
[0105] Primed in situ labeling (PRWS) is a molecular cytogenetic
technique that combines the high sensitivity of PCR with the
cellular or chromosome localization of fluorescent signals provided
by in situ hybridization. PRINS can be conducted by annealing
unlabelled PNA-DNA chimera primers to complementary target
sequences, followed by a DNA polymerase extension in the presence
of labelled dNTP. Preferably the labels are fluorescent dyes, so
that the extension products can be detected and/or measured by
fluorescence detection (Koch, 1991).
[0106] In one embodiment of the invention, the PNA-DNA chimera is
immobilized to a solid substrate through an ionic attraction,
affinity/receptor interaction, or covalent linkage. The solid
substrate may be particles, beads, membranes, frits, slides,
plates, micromachined chips, alkanethiol-gold layers, non-porous
surfaces, or other polynucleotide-immobilizing media. The solid
substrate material may be polystyrene, controlled-pore-glass,
silica gel, silica, polyacrylamide, magnetic beads, polyacrylate,
hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy,
and copolymers and grafts of such.
[0107] In this embodiment, the chimera may be physically
manipulated by automated means, e.g. assembling an addressable
array of multiple chimera, prior to primer-extension. Primer
extension reagents, including template, nucleotide
5'-triphosphates, and polymerase may be delivered in solution to
the location, well, vessel, or spot of the solid substrate bearing
the chimera. Primer extension may be conducted in such a
heterogeneous media. After extension is complete, all reagents in
solution may be conveniently removed by filtration, aspiration,
centrifugation, sedimentation, decanting, or magnetic pull-out of
magnetic particles. Alternatively, primer extension products may be
detached or released from the solid substrates in a pure state by
selective chemical, thermal, or enzymatic cleavage.
[0108] In another and similar embodiment, a template nucleic acid
may be immobilized on a solid substrate in the same configurations
and materials (supra). Primer extension reagents including PNA-DNA
chimera, nucleotide 5'-triphosphates, and polymerase may be
delivered in solution to the immobilized template and primer
extension conducted in a heterogeneous media. Primer extension
products can be conveniently separate from the template and
detected.
VI. EXAMPLES
[0109] The invention will be further clarified by a consideration
of the following examples, which are intended to be purely
exemplary of the present invention and not to limit its scope in
any way.
Example 1
Labelling of PNA-DNA Chimera
TAMRA and NTB Labeling
[0110] Labeling is performed with 5 mg of NHS ester of TAMRA or NTB
dissolved in 100 .mu.l DMF or NMP and 10 .mu.l DIEA added to the
support bound PNA-DNA chimera and allowed to react for 2 to 18
hours (typically overnight). The support is washed following the
labeling with DMF and subsequently DCM prior to cleavage.
CDPI Labeling
[0111] CDPI.sub.3 is attached to the chimera by three consecutive
couplings of Fmoc-CDPI (Lukhtanov, 1995) to give
CDPI.sub.3-labelled PNA-DNA chimera. The CDPI monomer unit,
1,2-dihydro-(3H)-pyrrolo[3,2-e]in- dole-7-carboxylate, protected
with Fmoc (5 mg, 0.012 mmole) is dissolved in 100 .mu.l NMP and
activated by 0.95 equivalents HATU (0.2M in DMF) and 2 equivalents
DIEA (0.4M in DMF). After one hour at room temperature, the
activated Fmoc-CDPI solution is added to the support bound chimera
and allowed to couple for another hour at room temperature. The
resin is washed following the coupling with 20 ml DMF. The Fmoc is
removed by treatment of the resin support with 1:4 piperidine:DMF
for 10 minutes at room temperature. This coupling and deprotection
cycle is repeated two additional times for a total of 3 manual
couplings to give CDPI.sub.3labelled PNA-DNA chimera.
Example 2
Primer Extension from DNA 38 mer Template with Klenow, Taq FS, and
no Enzyme Control
[0112]
5 Ac-TAG TTC - t (SEQ. ID NO.2) Ac-TAG TTC - ta (SEQ. ID NO.3)
Ac-TAG TTC - tag (SEQ. ID NO.4) Ac-TAG IIC - taga (SEQ. ID NO.5)
Ac-TAG TTC T - ag (SEQ. ID NO.9) Ac-TAG TTC T - aga (SEQ. ID NO.10)
Ac-TAG TTC T - agac (SEQ. ID NO.11) UPPER CASE = PNA, lower case =
DNA. All chimera above have amide linkage. Amino terminus of PNA is
acetylated (Ac).
[0113] 2.1. Annealing of Primers and Templates
[0114] PNA-DNA chimera, PNA, and DNA primers (FIGS. 7-11) were
annealed to the synthetic 38-mer DNA oligonucleotide templates:
6 (SEQ. ID. NO.8) 5' CGC TCA ACA CAT AGC ATG GTC TAG AAC TAA GCC
TGG AA 3' (SEQ. ID. NO.12) 5' CGC TCA ACA CAT AGC ATG GTC CAG AAC
TAA GCC TGG AA 3' (SEQ. ID. NO.13) 5' CGC TCA ACA CAT AGC ATG GCC
TAG AAC TAA GCC TGG AA 3'
[0115] where bold, underlined bases indicate mismatch bases. The
mixtures were heated to 95.degree. C. and slowly cooled to
37.degree. C. during one hour in a thermocycler instrument (PE
GeneAmp PCR System 9700, PE Biosystems).
[0116] 2.2. Polymerase Extension Reaction with Non-thermostable DNA
Polymerases
[0117] PNA-DNA chimera and DNA primers were extended from their
respective 3'-OH ends for 2 to 16 h at 37.degree. C. with 2.5-50
units of DNA polymerase, e.g. Klenow, T4, or Bst DNA polymerase,
and primer-extension buffer in 25 to 100 .mu.l total volume. In the
case of Klenow, for example, 0.1 to 1 mM each
nucleotide-5'-triphosphate and 1.times.EcoPol buffer containing 10
mM Tris-HCl (pH 7.5), 5 mM MgCl.sub.2, and 7.5 mM dithiothreitol
were added into each reaction.
[0118] 2.3. Primer Extension Reaction with Thermostable DNA
Polymerases
[0119] A total of 50 .mu.l of primer extension mixture generally
includes 2.5 to 25 U thermostable polymerase (i.e. AmpliTaq Gold),
1.times.PCR buffer II containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3,
200 to 500 mM of each dNTP, and 2 to 4 mM of MgCl.sub.2. Primer
extension is performed for 25-40 rounds of thermal cycling in a
program (10 min at 95.degree. C. once, then 0.5 min at 95.degree.
C., 1 to 5 min at 37 to 67.degree. C., 1 to 10 min at 60 to
72.degree. C. for cycling).
[0120] 2.4. Electrophoresis and Visualization
[0121] After incubation, reaction product was placed on ice or at
4.degree. C. for a short period. Typically, 5 to 25 pmol of the
extended product was mixed with a final concentration of
1.times.loading buffer (45 mM Tris base, 45 mM boric acid, 0.4 mM
EDTA, 3% Ficoll, 0.02% bromophenol blue, 0.02% xylene cyanol) and
denatured at 95.degree. C. for 10 to 20 min. The sample is loaded
into a 10 to 15% denaturing PAGE gel and run in 1.times.TBE (89 mM
Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.3) at 100 to 160 V,
70.degree. C. for 25 to 60 min. The extended product was visualized
by staining the gel with SYBR-Green (Molecular Probes, Eugene,
Oreg.) in a volume of 40 to 120 ml in 1.times.TBE for 10 to 30 min.
The image was captured in an ChemImaging 2000 gel documentation
system.
[0122] In the case of Klenow polymerase, for example, reaction
conditions are 25.degree. C. and 10 mM Tris-HCl pH 7.5, 5 mini
MgCl.sub.2, 7.5 mM dithiothreitol, 1 mM each nucleotide
5'-triphosphate, 0.1 to 100 pmoles chimera, 0.1 to 100 pmoles
template, and 0.1 to 10 units Klenow enzyme in 5 to 500 .mu.l total
volume.
[0123] A Taq polymerase primer extension reaction may be conducted
at 72-80.degree. C. and contain 10 mM KCl, 20 mM Tns-HCl pH 8.8, 10
mM (NH.sub.4).sub.2SO.sub.4, 2 mM MgSO.sub.4, 0.1% Triton X-100
detergent, 1 mM each nucleotide 5'-triphosphate, 0.1 to 100 pmoles
chimera, 0.1 to 100 pmoles template, and 0.1 to 10 units Taq
polymerase enzyme in 5 to 500 .mu.l total volume.
[0124] 2.5. MALDI-TOF Analysis
[0125] Mass spectra is acquired on a MALDI-TOF MS (Voyager DE)
workstation. Desalted samples are mixed 1:1 with matrix solution
consisting of 50 mg/ml 3-hydroxy picolinic acid, 50 mM ammonium
citrate, and 30% acetonitrile, and is spotted onto a sample plate.
Time-of-flight data from 20 to 50 individual laser pulses are
recorded and averaged on a transient digitizer, after which the
averaged spectra are automatically converted to mass by data
processing software.
Example 3
RT-PCR Murine Xist Gene
[0126]
7 P1 Ac-TA GGT CCC GGC ttta (SEQ. ID NO.14) P2 Ac-TA GGT CCC GGC t
(SEQ. ID NO.15) D1 AAC AGT TA GGT CCC GGC TTT (SEQ. ID NO.16) D2
ACT GGG ATG CAA AGA GCA TT (SEQ. ID NO.17) D3 TGC CTG GGA TAA AAG
CAA AG (SEQ. ID NO.18)
[0127] Total RNA was isolated by using the guanidinium thiocyanate
method from kidneys of male and female mice (Chirgwin, 1979).
Reverse transcription was conducted on 0.5 to 1.5 .mu.g total RNA
samples with 10 pmoles of Xist-specific primers including DNA RT
primer D1 or PNA-DNA chimera RT primers P1 and P2, respectively,
0.2-1 mM each of dATP, dGTP, dCTP, dTTP, 10 to 20 .mu.l RT reaction
buffer (10 mM Tris-HCl, pH 8.3, 90 mM KCl), and 2 to 10 U
recombinant Thermals thermophilus (rTth) DNA polymerase. The
solution was incubated for 10 min at 65.degree. C. followed by 60
mm at 60.degree. C. The samples were PCR amplified (30 s at
94.degree. C., 30 s at 55.degree. C. and 30s at 65.degree.C.) in
1.times.chelating buffer [5% (v/v) glycerol, 10 mM Tris-HCl, pH
8.3, 0.05% Tween 20, 0.75 mM EDTA] with 2 to 10 U rTth DNA
polymerase and 20 pmoles of each primer (D2 and D3). PCR products
were separated and analyzed by 1 to 3% agarose gel electrophoresis
with SYBR-Green staining (FIG. 13). The P2 PNA-DNA chimera, with
four 2'-deoxynucleotides, was effective in producing an amplifiable
copy of mouse Xist gene, whereas the P1 chimera, with only one
2'-deoxynucleotide, was not.
[0128] In the case of M-MuLV reverse transcriptase, altered
reaction conditions were 37 .degree. C. and 50 mM Tris-HCl pH 8.3,
8 mM MgCl.sub.2, 10 mM dithiothreitol, 1 mM each dNTP, 0.1 to 100
pmoles chimera primer, 0.1 to 100 pmoles template, and 0.1 to 10 U
M-MuLV enzyme in 5 to 500 .mu.l total volume.
Example 4
SNP Detection
[0129] A nested PCR is generally recommended for genomic targets.
Briefly, 5 pmoles PCR primers flanking the target sequences are
subject to 10 rounds of thermal cycling (30 s at 94.degree. C., 30
s at 55-70.degree. C.) in 25 .mu.l of reaction buffer comprising
1.times.Taq buffer described previously, 400 .mu.M of each dNTP, 4
mM MgCl.sub.2, 20-50 ng human genomic DNA, 0.5 to 5 U Taq DNA
polymerase or other thermostable enzymes. After 50 pmoles specific
pairs of DNA primers are added, the mixture is thermal cycled for
an additional 15-20 rounds to amplify all loci.
Multiplex SNP Extension Assay
[0130] To 20 .mu.l PCR mixture is added 1 U each of shrimp alkaline
phosphatase and 10 U exonuclease I. The mixture is incubated for 15
min at 37.degree. C. followed by 15 min at 85.degree. C. Then, 20
.mu.l of a mixture containing 25-100 .mu.M each ddNTP. 2 mM
MgCl.sub.2, 1 to 5 U Taq polymerase, 1.times.PCR buffer, and 20-50
pmoles of each PNA-DNA chimera primer is added subsequently. The
resultant mixture is subjected to 25-35 rounds of thermal cycling
(30 s at 94.degree. C., 30 s at 37 to 67.degree. C., 20-90 s at
70.degree. C.). Desalting 10 to 50 .mu.l of the reaction mixture is
performed by absorption/elution using ZipTip or 96-well plate
packed with small quantities of POROS 50 R1, R2, or R3
chromatography media.
Example 5
Fluorescence Detection of Primer Extension of PNA-DNA Chimera with
TAN IRA-dUTP
[0131] PNA-DNA chimera primer (SEQ. ED NO. 4) was extended with 3
different mixtures of nucleotides (a., b., c.) after annealing to
5' biotin DNA 38 nt template (SEQ. ID NO. 8) with a variety of
polymerases (FIG. 15). The biotin label serves to enable capture
affinity of duplex extension products or recovery of template by
binding to avidin, e.g. immobilized strepavidin. Polyacrylamide
(15%) gel electrophoresis under denaturing conditions with
fluorescence detection (no staining) showed only the expected
fluorescence in reactions (b) with TAMRA-dUTP. The dark bands at 15
and 20 bp correspond to TAMRA-dUTP and a dimer artifact,
respectively. Full length extension product is apparent in
reactions employing (left to right) Klenow, AmpliTaq, TaqGold,
Vent, Stoffel fragment, and Sequenase as a faint band migrating at
the rate of a 30 bp DNA duplex. These results demonstrate the
incorporation of a labelled nucleotide, e.g. TAMRA-dUTP, with a
range of polymerases.
Example 6
Primed In Situ Labeling (PRINS)--Chromosome Labelling by PNA-DNA
Chimera Primer Extension with TAMRA-dUTP
[0132] A reaction mixture containing 1 -3 .mu.M PNA-DNA chimera
primer, 100 to 200 .mu.M of each dATP, dCTP, dGTP, 20 .mu.M dTTP,
20 .mu.M of FAM-12-dUTP, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1-5 mM
MgCl.sub.2, 0.01% bovine serum albumin, and 2 to 10 U Taq DNA
polymerase is prepared to a final volume of 50 .mu.l. A total of 20
to 30 .mu.l reaction mixture is placed on each slide. The slide is
incubated on a programmable temperature cycler (PE Ampli2000). The
program consists of 15-30 min at 50-65.degree. C. for annealing and
30 to 120 min at 72.degree. C. for extension. The reaction is
stopped by immersing the slides in 50 mM NaCl, 50 mM EDTA, pH 8 at
72.degree. C. for 5 min. After incubation, the slides are washed
three times with 70% formamide/10 mM Tris pH 7.2 for 10 min and
with 0.05 M Tris/0.15 M NaCl/0.05% Tween-20 pH 7.5 for 5 min. The
slides are then dehydrated in an ethanol series and air dried in
the dark. Chromosomes are counterstained with either 0.1 .mu.g/ml
of 4,6-diamidino-2-phenylindole-dehydrochloride (DAPI) in antifade
or 0.6 .mu.g/ml of propidium iodide (Oncor, Gaitherburg, Md.).
Example 7
DNA Sequencing with PNA-DNA Chimera Primer and Fluorescent
Dye-labelled Terminating Nucleotide 5'-triphosphates
[0133] Rhodamine labelled, 2',3'-dideoxynucleotides and PNA-DNA
chimera primer:
Ac-ACG ACG GCC agt 3' (SEQ. ID NO. 19)
[0134] are used to label DNA fragments in chain termination
sequencing on an Applied Biosystems 310 Genetic Analyzer. The
template nucleic acid, pGEM, (0.4 pmoles) was annealed with the
primer (0.8 pmoles) and primer-extension reagents comprising 2
.mu.l buffer (400 mM Tris-HCl, 10 mM MlgCl.sub.2, pH 9.0.), 2 .mu.l
of a deoxynucleotide/labelled dideoxynucleotide mixture, and 2
.mu.l of AmpliTaq DNA polymerase FS enzyme (5 Units/.mu.l). The FS
enzyme is a recombinant Thermus aquaticus DNA polymerase having two
point mutations--G46D and F667Y. The protocol is provided in the
ABI PRISM.TM. Dye Terminator Cycle Sequencing Core Kit Manual (PE
Biosystems). The reaction is then thermocycled using the following
exemplary program: denaturation at 98.degree. C. for 5 s followed
by repeated cycles of: 96.degree. C. for 5 s, 55.degree. C. for 40
s, and 68.degree. C. for 1 min. This cycle is repeated
approximately 15 times.
[0135] The nucleotide mixture consists of dNTP: 2 mM each dATP,
dCTP, 7-deaza-dGTP and dTTP, and labelled ddNTP: 9.0 .mu.M
5R6G-ddATP, 2.7 .mu.M 5R110-ddGTP, 54 .mu.M 6ROX-ddCTP, and 216
.mu.M 6TMR-ddTTP.
[0136] The primer extension sequencing reactions can be conducted
in 0.5 ml tubes adapted for a thermal cycling reaction period in a
thermal cycler, e.g. Perkin-Elmer 480 DNA Thermal Cycler (PE
Biosystems). Reaction volumes may be 20 .mu.l, including 15 .mu.l
of the above reaction premix, a variable amount of fluorescent
dye-labeled terminator, and a sufficient volume of water to brine
the total reaction volume up to 20 .mu.L. From 1 to 1000 pmol of
the dye terminator can be added to each reaction. Mineral oil (30
.mu.l) is added to the top of each reaction to prevent evaporation.
Reactions are thermocycled as follows: 96.degree. C. for 30 sec,
50.degree. C. for 15 sec, and 60.degree. C. for 4 min, for 25
cycles; followed by a 4.degree. C. hold cycle.
[0137] Reactions are purified on Centri-Sep spin columns according
to manufacturer's instructions (Princeton Separations). After the
column is hydrated with 0.8 mL deionized water for at least 30
minutes at room temperature, inspected to determine that no bubbles
are trapped in the gel material, the upper and lower end caps of
the column are removed, and the column is allowed to drain by
gravity. The column is then inserted into the wash tubes provided
in the kit and centrifuged in a variable speed microcentrifuge at
13,000.times.g, for 2 minutes, removed from the wash tube, and
inserted into a sample collection tube The reaction mixture is
carefully removed from under the oil and loaded onto the gel
material. Loaded columns are centrifuged to elute the samples which
are then dried in a vacuum centrifuge.
[0138] Prior to loading onto a sequencing gel, the dried samples
are resuspended in 25 .mu.l of Template Suppression Reagent (PE
Biosystems), vortexed, heated to 95.degree. C. for 2 minutes,
cooled on ice, vortexed again, and centrifuged. A 10 .mu.l aliquot
of the resuspended sample is transferred to sample vials for
electrophoresis on the PE ABI PRISM.TM. 310 Genetic Analyzer (PE
Biosystems) with sieving polymers including nucleic acid
denaturants, and capillaries specially adapted for DNA sequence
analysis. Samples are electrokinetically injected onto the
capillary for 30 s at 2.5 kV, and run for 2 hr at 10 to 12.2 kV
with the outside wall of the capillary maintained at 42.degree.
C.
[0139] All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
[0140] Although only a few embodiments have been described in
detail above, those having ordinary skill in the relevant arts will
clearly understand that many modifications are possible in the
preferred embodiment without departing from the teachings thereof.
All such modifications are intended to be encompassed within the
following claims.
VII. References
[0141] Andrus, A. "Chemical methods for 5' non-isotopic labelling
of PCR probes and primers" (1995) in PCR 2: A Practical Approach,
Oxford University Press, Oxford, pp. 39-54.
[0142] Beaucage, S. and Jyer, R. "Advances in the synthesis of
oligonucleotides by the phosphoramidite approach", Tetrahedron
48:2223-2311 (1992).
[0143] Bergot, B., Chakerian, V., Connell, C., Eadie, J., Fung, S.,
Hershey, N., Lee, L., Menchen, S. and Woo, S. "Spectrally
resolvable rhodamine dyes for nucleic acid sequence determination",
U.S. Pat. No. 5,366,860, issued Nov. 22, 1994.
[0144] Blackburn, G. and Gait, M. Eds. "DNA and RNA structure" in
Nucleic Acids in Chemistry and Biology, 2nd Edition. (1996) Oxford
University Press.
[0145] Breipohl, G., Will, D. W., Peyman, A. & Uhlmann, E.
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