U.S. patent application number 14/809635 was filed with the patent office on 2015-11-12 for reducing template independent primer extension and threshold time for loop mediated isothermal amplification.
This patent application is currently assigned to NEW ENGLAND BIOLABS, INC.. The applicant listed for this patent is NEW ENGLAND BIOLABS, INC.. Invention is credited to Thomas C. Evans, JR., Nathan Tanner.
Application Number | 20150322472 14/809635 |
Document ID | / |
Family ID | 48281014 |
Filed Date | 2015-11-12 |
United States Patent
Application |
20150322472 |
Kind Code |
A1 |
Tanner; Nathan ; et
al. |
November 12, 2015 |
Reducing Template Independent Primer Extension and Threshold Time
for Loop Mediated Isothermal Amplification
Abstract
Compositions and methods are provided for loop mediated
isothermal amplification in which single stranded binding proteins
are shown to protect primers from non-specific extension and to
stimulate the rate of threshold amplification.
Inventors: |
Tanner; Nathan; (Peabody,
MA) ; Evans, JR.; Thomas C.; (Topsfield, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NEW ENGLAND BIOLABS, INC. |
Ipswich |
MA |
US |
|
|
Assignee: |
NEW ENGLAND BIOLABS, INC.
Ipswich
MA
|
Family ID: |
48281014 |
Appl. No.: |
14/809635 |
Filed: |
July 27, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13671123 |
Nov 7, 2012 |
9121046 |
|
|
14809635 |
|
|
|
|
61560518 |
Nov 16, 2011 |
|
|
|
Current U.S.
Class: |
435/194 |
Current CPC
Class: |
C12Q 1/6848 20130101;
C12P 19/34 20130101; C12Q 1/6848 20130101; C12Q 2522/101 20130101;
C12Q 2531/119 20130101; C12Q 2527/101 20130101; C12Q 2527/125
20130101 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Claims
1. A preparation, comprising: a single stranded binding protein
(SSB); a thermostable polymerase; at least four oligonucleotide
primers, and a buffer.
2. A preparation according to claim 1, wherein the buffer has a pH
in the range of pH6-pH9, and optionally a stabilization agent
selected from the group consisting of BSA, glycerol and
detergent.
3. A preparation according to claim 1 wherein the buffer comprises
a monovalent salt having a concentration in the range of 0-500
mM.
4. A preparation according to claim 1 wherein the buffer comprises
a divalent metal cation having a concentration of 0.5 mM-10 mM.
5. A preparation according to claim 1, wherein the buffer has a pH
in the range of pH6-pH9, a monovalent salt having a concentration
in the range of 0-500 mM, a divalent metal cation having a
concentration of 0.5 mM-10 mM and optionally a stabilization agent
selected from the group consisting of BSA, glycerol and
detergent.
6. A preparation according to claim 1, wherein the SSB is an
extreme thermophile single strand binding protein (ET SSB).
7. A preparation according to claim 1, wherein the thermostable
polymerase has strand displacement activity and is active at
temperatures of greater than 50.degree. C.
Description
CROSS REFEREENCE
[0001] This application is a divisional of U.S. application Ser.
No. 13/671,123 filed Nov. 7, 2012 which claims right of priority to
U.S. Provisional Application No. 61/560,518 filed Nov. 16,
2011.
BACKGROUND
[0002] Amplification of target nucleic acids is a fundamental
method in modern molecular biology and diagnostics. Factors that
adversely affect the outcome of amplification reactions include:
extension of primers due to non-specific template annealing during
amplification, resulting in false positives (Schlotterer and Tautz,
Nucleic Acids Research, 20 (2):211-215 (1992); Ogata and Miura,
Nucleic Acids Research, 26(20):4657-4661 (1998); Brukner, et al.
Analytical Biochemistry, 339:345-347 (2005)); reduced amplification
reaction efficiency and rate due to primer or template secondary
structure; and variability of amplification due to primer dimer
formation. The effects of these factors are enhanced by room
temperature (RT) incubation of complete reaction mixtures prior to
placement at specified reaction temperature. This would occur, for
example, when large numbers of samples are prepared at one time
necessitating a certain amount of sample incubation at RT.
Therefore, high-throughput and diagnostic applications are often
negatively impacted by reaction set-up at RT. This is a significant
issue for molecular diagnostic applications, which, demands a high
level of consistency and accuracy.
[0003] Various amplification methods are currently utilized in
molecular diagnostics. A popular isothermal amplification
diagnostic method is loop-mediated isothermal amplification (LAMP)
(Notomi, et al. Nucleic Acids Research, 28(12):e63 (2000)).
Typically, LAMP employs a DNA polymerase and a set of four to six
synthetic primers that recognize a total of six distinct sequences
on the target DNA. Recognizing six distinct sequences makes LAMP
extremely specific for a target sequence. Despite the specificity
of LAMP, it is adversely affected by unwanted, non-specific primer
extension reactions during reaction set-up at RT.
SUMMARY
[0004] In general in one aspect, a preparation includes a SSB; a
thermostable polymerase; at least four oligonucleotide primers, and
a buffer.
[0005] In another aspect, the buffer in the preparation has a pH in
the range of pH6-pH9, a monovalent salt having a concentration in
the range of 0-500 mM, a divalent metal cation having a
concentration of 0.5 mM-10 mM and optionally a stabilization agent
selected from the group consisting of BSA, glycerol and
detergent.
[0006] In another aspect, the SSB in the preparation is an extreme
thermophile single strand binding protein (ET SSB) (New England
Biolabs, Ipswich, Mass.).
[0007] In another aspect, the thermostable polymerase in the
preparation has strand displacement activity and is active at
temperatures of greater than 50.degree. C.
[0008] In another aspect, the preparation is used in a method for
amplifying a nucleic acid, which includes adding to the
preparation, dNTPs and template nucleic acid; performing LAMP; and
obtaining amplified template DNA.
[0009] In general in one aspect, a method for inhibiting primer
extension of a primer in an amplification reaction, includes:
combining a SSB with a thermostable polymerase, at least four
primers and a template nucleic acid in a reaction buffer at a first
temperature; performing a LAMP reaction at a second temperature
which is greater than the first temperature; and determining the
inhibition with respect to the same mixture without the SSB.
[0010] In another aspect, the method includes obtaining an
increased rate of LAMP of the template DNA.
[0011] In general in one aspect, a method for obtaining an increase
in a rate of LAMP, includes combining a SSB with a thermostable
polymerase, at least four primers and a template nucleic acid in a
reaction buffer at a first temperature; and immediately or after a
lag time at a temperature above 4.degree. C. but below 70.degree.
C., performing a LAMP reaction at a second temperature, wherein the
increase is determined with respect to the same mixture without the
SSB.
[0012] In another aspect, the increase in the rate of amplification
is measured by time taken to reach threshold amplification is more
than 25%.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A-B show the effect on amplification efficiencies
when a LAMP reaction mixture (Notomi, et al. (2000)) was tested
either immediately after removal of the sample from incubation on
ice (solid line); or after a 2 hour pre-incubation of the sample at
25.degree. C. before moving the sample to the 65.degree. C.
reaction temperature (dashed line).
[0014] After the pre-incubation, LAMP amplification was performed
and the threshold time required to produce a threshold amount of
fluorescent signal from a DNA intercalating dye was determined (see
also Examples 1 and 2).
[0015] The results in FIGS. 1A and 1B showed that the reaction time
to threshold signal for LAMP increased from 25 minutes for samples
subjected to 0 hour pre-incubation at 25.degree. C. to 40 minutes
for the samples that had been subjected to a 2 hour pre-incubation
at 25.degree. C. for a reaction mixture containing Bst DNA
polymerase, large fragment, and primers.
[0016] FIG. 1A shows results for reactions that contained LAMP
primers, where the primers were forward internal primer (FIP),
backward internal primer (BIP), forward external primer (F3) and
backward external primer (B3). Results are shown for samples that
contained Bst DNA polymerase, large fragment (Bst) only; Bst
polymerase plus all primers; and Bst polymerase plus all primers
plus lambda DNA template (temp). The deleterious effect of
pre-incubation at 25.degree. C. was observed for reactions that
contained Bst polymerase and primers.
[0017] FIG. 1B shows the result of pre-incubation of Bst polymerase
with specified LAMP primers (FIP; BIP; F3; B3; FIP+BIP; F3+B3; no
primers or all primers) at 25.degree. C. prior to the amplification
reaction at 65.degree. C. The deleterious effect of pre-incubation
at 25.degree. C. was observed for reactions that contained Bst
polymerase and primers
[0018] FIGS. 2A-B show capillary electrophoresis (CE) analysis of
primer extension reactions that occurred at 25.degree. C. The
estimated number of nucleotides based on retention time in the CE
is provided on the x-axis and the peak height in relative
fluorescence units is given on the y-axis of each electropherogram.
Primer extension was measured for individual primers in the
presence or absence of an ET SSB. The results show that ET SSB is
capable of protecting primers from undesirable extension reactions
in the presence of a DNA polymerase, PoID, during LAMP reaction
setup (2 hours at 25.degree. C.). Template DNA was not included in
the reaction. The observed protection was not primer specific as
demonstrated in FIG. 2A, which utilized the BIP primer from the
primer set described above and FIG. 2B which utilized a BIP from a
different LAMP primer set.
[0019] The LAMP primers were incubated at 1.6 .mu.M in reaction
buffer as follows, with all reactions performed in 25 .mu.L
volumes: [0020] (i) BIP primer, buffer, 2 hour incubation at
25.degree. C. no ET SSB. [0021] (ii) BIP primer, 10U PoID
polymerase, buffer, 2 hour incubation at 25.degree. C., no ET SSB.
[0022] (iii) BIP primer, 10U PoID polymerase, buffer, 2 hour
incubation at 25.degree. C., 1 .mu.g ET SSB. [0023] (iv) BIP
primer, 10U PoID polymerase, buffer, 2 hour incubation at
25.degree. C., 2 .mu.g ET SSB.
[0024] Subsequent to pre-incubation, the 1.6 .mu.M samples were
diluted to 5 nM and analyzed using CE. A CE peak can be seen in (i)
indicating the unmodified primer, which in (ii) runs significantly
larger, corresponding to the extension products of the
fluorescently-labeled primer due to DNA polymerase activity. These
extension peaks became diminished in the presence of the increasing
amounts of SSB (iii and iv), indicating inhibition of extension of
the primers at RT.
[0025] FIG. 2A shows data obtained using a 5'-FAM labeled lambda1
BIP primer from LAMP primer set used in FIGS. 1A-B, 3, and 4A-B
(5'-6FAM-GAGAGAATTTGTACCACCTCCCACCGGGCACATAGCAGTCCTAGGGACAGT, IDT)
(SEQ ID NO:1).
[0026] FIG. 2B shows data obtained using a 5'-FAM labeled lambda 2
BIP primer from a different set of LAMP primers (5'-6FAM -3')
CAGGACGCTGTGGCATTGCAGATCATAGGTAAAGCGCCACGC (SEQ ID NO:2).
[0027] FIG. 3 shows the results of an assay for determining optimal
amounts of ET SSB (1-3 .mu.g) in the reaction mixture.
[0028] A single sample was divided into two aliquots. One aliquot
was amplified immediately after removal from setup on ice (solid
line). The second aliquot was amplified after the sample was
pre-incubated at RT (25.degree. C.) for 2 hours (dashed line). An
increase in the rate of the amplification reaction as measured by a
reduction in threshold time was observed, with the greatest
stimulation observed above 1 .mu.g ET SSB (reactions performed in
25 A volume). The lag time between immediate (solid) and 2 hour
incubated (dashed) samples also decreased, with an approximately
100% (20 minutes) delay observed without ET SSB, but no delay
observed with 2 .mu.g ET SSB. This demonstrates that ET SSB added
to primers during the pre-incubation at RT enhanced amplification
rates.
[0029] FIGS. 4A-B show the beneficial effect of ET SSB on threshold
time for amplification and protection of primers from non-template
extension.
[0030] FIG. 4A shows percent stimulation in threshold time for LAMP
of target DNA when 0-3 .mu.g ET SSB was added to primers prior to
amplification (no incubation time). Concentrations of 1.5-3.0 .mu.g
ET SSB added to 25 .mu.L reactions resulted in greater than 50%
stimulation of amplification as measured by decreasing time to
amplification threshold.
[0031] FIG. 4B shows the effect of 0-3 .mu.g ET SSB on protection
of primers from non-template extension due to pre-incubation at RT
prior to performing LAMP reactions. 100% protection of the primers
was achieved by use of 1-3 .mu.g ET SSB added to 25 .mu.L
reactions. Protection was measured by difference between threshold
times of the sample with no pre-incubation and the sample
pre-incubated at RT for 2 hours, with no threshold time delay
defined as 100% protection.
DESCRIPTION OF EMBODIMENTS
[0032] The problem of variability that arises from sample handling
prior to amplification has been solved by the compositions and
methods described herein. Primers combined with SSB that are
allowed to stand at RT in the presence of polymerase are protected
from undesired DNA polymerase dependent replication or extension in
the absence of template DNA otherwise observed at temperatures
lower than the amplification reaction temperature. This protection
is not primer sequence dependent. The protective effect of SSBs
results in one or more of the following benefits: reduced
variability in threshold times for amplification, shorter times to
reach threshold amplification and reduced lag time before
amplification is initiated.
[0033] Generally, a stimulation of amplification reaction
efficiency can decrease time to reach a defined threshold level of
amplification, minimizing required reaction and diagnostic times.
The beneficial effect of SSBs is observed when the time to reach
the defined threshold is decreased. An increase in the rate of LAMP
has been identified when SSBs are added to a buffer in which the
reaction is subsequently performed. The increase in rate is
measured by the time required to achieve a threshold yield of
amplicon. The observed increase is at least 50% when SSB is added
to a polymerase primer mix at, for example, one to two molar
equivalents of the DNA primers or for example 0.5-10 .mu.g, 1.0-5
.mu.g or 1.5-3.0 .mu.g of SSB.
[0034] Threshold times may be based on sufficient amplification to
produce a detectable signal, for example, a fluorescent signal with
an intercalating dye on a real time fluorimeter in the range of
100-500,000 RFU, preferably at least 1000 RFU. Alternatively,
turbidity methods can be used where threshold is defined as dT/dt
greater than 0.1.
[0035] In addition to stimulation of reaction time efficiency,
protection from non-template primer extension is also provided by
the SSB. While the protection from non-template primer extension is
not primer dependent and is observed for primers regardless of
sequence, some variation in the extent of protection may occur.
However, in all cases, the benefit is significant. The protection
can range from 25% to 100% where 100% protection is equivalent to
the optimal efficiency of amplification when a sample is removed
from a 4.degree. C. environment and immediately amplified without
any RT incubation and 0% is the protection against non-template
primer extension afforded after a pre-incubation of primers with
polymerase for 2 hours at 25.degree. C. in the absence of SSB (see
FIGS. 2A-B and 4A-B).
[0036] Protection can be achieved when an SSB is added to a
polymerase primer mix at, for example, one to two molar equivalents
of the DNA primers or for example 0.5-10 .mu.g, 1.0-5 .mu.g or
1.5-3.0 .mu.g of SSB in a 25-50 .mu.l reaction.
[0037] The addition of SSB protects against the negative
consequences of RT setup of amplification reactions prior to
raising the temperature to initiate amplification as the SSB
prevents primer extension to an extent of at least 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. The
pre-incubation time although exemplified as 2 hours at RT could be
as little as 5 minutes or as much as 24 hours. The pre-incubation
temperature although exemplified by 25.degree. C. is intended to
include any temperature greater than 4.degree. C. and less than
50.degree. C.
[0038] The present methods and compositions can be used for a hot
start amplification in which any non-template primer extension is
blocked at RT in the presence of SSB prior to raising the
temperature to 50.degree. C-70.degree. C. in an isothermal reaction
such as LAMP.
[0039] As described above, the protection of primers from extension
by SSB under these conditions gives rise to stimulation of
amplification efficiency and reduced variability in amplification
reactions and enhances reaction performance.
[0040] Examples of SSBs known in the art that may be used in the
present methods include: bacterial SSBs (e.g. E. coli SSB) and
phage SSBs (T4 gp32, T7 gp2.5) (Hamdan and Richardson, Annual
Review of Biochemistry, 78:205-243 (2009)). SSBs from eukaryotic
organisms (e.g. RPA) have similar mechanisms of action and
interaction in DNA replication and repair processes (Richard, et
al. Critical Reviews in Biochemistry and Molecular Biology, 44
(2-3):98-116, (2009)) and may be used herein. While a thermostable
SSB is exemplified here, this is not intended to be limiting.
[0041] ET SSB is a 16 kDa single-stranded DNA binding protein which
is fully active after 60 minutes at 95.degree. C. and can
destabilize secondary structure, and improve DNA polymerase
activity (Richard, et al. Nucleic Acids Research, 32 (3):1065-1074,
(2004)). The ET SSB can be used for hot start amplification and for
PCR, RT-PCR, HDA, RCA, sequencing, and isothermal amplification
reactions.
[0042] Thermostable polymerases for use in LAMP include PoID; Bst
DNA polymerase large fragment; or mutants thereof; or
WarmStart.RTM. Bst 2.0 DNA polymerase (New England Biolabs,
Ipswich, Mass.) (Notomi et al. (2000); Tanner, et al.,
BioTechniques, 53:81-89, (2012)).
TABLE-US-00001 TABLE 1 Examples of oligonucleotides (LAMP primers)
showing similar protection and threshold stimulation to that shown
in Figures 3, 4A and 4B for SEQ ID NOs: 1 and 2. Target Primer
Sequence lambda 1 FIP
CAGCCAGCCGCAGCACGTTCGCTCATAGGAGATATGGTAGAGCCGC (SEQ ID NO: 3) BIP
GAGAGAATTTGTACCACCTCCCACCGGGCACATAGCAGTCCTAGGGACAGT (SEQ ID NO: 1)
F3 GGCTTGGCTCTGCTAACACGTT (SEQ ID NO: 4) B3 GGACGTTTGTAATGTCCGCTCC
(SEQ ID NO: 5) Loop F CTGCATACGACGTGTCT (SEQ ID NO: 6) Loop B
ACCATCTATGACTGTACGCC (SEQ ID NO: 7) lambda 2 FIP
AGGCCAAGCTGCTTGCGGTAGCCGGACGCTACCAGCTTCT (SEQ ID NO: 8) BIP
CAGGACGCTGTGGCATTGCAGATCATAGGTAAAGCGCCACGC (SEQ ID NO: 2) F3
AAAACTCAAATCAACAGGCG (SEQ ID NO: 9) B3 GACGGATATCACCACGATCA (SEQ ID
NO: 10) Loop F GCATCCCACCAACGGGAA (SEQ ID NO: 11) Loop B
CAGATTAAGGA (SEQ ID NO: 12) lambda C FIP
CGAACTGTTTCGGGATTGCATTCTGGAACTCCAACCATCGCA (SEQ ID NO: 13) BIP
GGAGCCTGCATAACGGTTTCGTCGACTCAATGCTCTTACCTGT (SEQ ID NO: 14) F3
GTTGGTCACTTCGACGTATCG (SEQ ID NO: 15) B3 GCTCGCCGACTCTTCACGAT (SEQ
ID NO: 16) Loop F TTTGCAGACCTCTCTGCC (SEQ ID NO: 17) Loop B
GGATTTTTTATATCTGCACA (SEQ ID NO: 18) E. coli dnaE FIP
CTGCCCCGACGATAGGCTTAATCGTGGTCTGGTGAAGTTCTACGG (SEQ ID NO: 19) BIP
TCCAGTGCGACCTGCTGGGTGGGTATTGTTCGCCGCCAGTAC (SEQ ID NO: 20) F3
GATCACCGATTTCACCAACC (SEQ ID NO: 21) B3 CTTTTGAGATCAGCAACGTCAG (SEQ
ID NO: 22) Loop F TGCGCCATGTCCCGCT (SEQ ID NO: 23) Loop B
TGAGTTAACCCACCTGACG (SEQ ID NO: 24) C. elegans FIP
TGTTAAGGCGGACTGTGTTCGTCAAACCGCAACGAGACAGTCT lec-6 (SEQ ID NO: 25)
BIP CCGAGATAATTCCACCGTTGGATCCATTCCAGCAGAACAAGAT (SEQ ID NO: 26) F3
GATGTCACGAAAAATTCCCTC (SEQ ID NO: 27) B3 GCAATCCGAGGATCGTCAC (SEQ
ID NO: 28) Loop F TGCAAAGCACGTGGTGCC (SEQ ID NO: 29) Loop B
ACACAAACTCCAGAGTGTAG (SEQ ID NO: 30) C. elegans FIP
CTCTGTGAACGGTCATCACCTCGATGGCTTGAACCGATTGGTATGG lec-10a (SEQ ID NO:
31) BIP CTTACATGGTAATATCCAGCGTGCCACTTCACCACTCGGAGCAC (SEQ ID NO:
32) F3 GAACGTCTCCCTTCAATCC (SEQ ID NO: 33) B3 GGACCAGAAATCCGTCACA
(SEQ ID NO: 34) Loop F CCGACTACCCACATCGTTAC (SEQ ID NO: 35) Loop B
ACCTTGATGCTAAGGTGGAA (SEQ ID NO: 36) C. elegans FIP
GATTCCACTTCCAACGTCGTTG-CATAGGCATTGTATCCAGAGTG lec-10b (SEQ ID NO:
37) BIP CGAAGTGAACCTTGTCAACATGAGACTACCCACATCGTTACC (SEQ ID NO: 38)
F3 AGCAACATAGGTTTCAGTTC (SEQ ID NO: 39) B3 CTGTGAACGGTCATCACC (SEQ
ID NO: 40) Loop F ACGGACATGTCGATCATGGA (SEQ ID NO: 41) Loop B
CGTCTCCCTTCAATCCGATGGC (SEQ ID NO: 42) pUC19 AmpR FIP
ATGGGGGATCATGTAACTCGCCTCGTCGTTTGGTATGGCTTC (SEQ ID NO: 43) BIP
AAGCGGTTAGCTCCTTCGGTCTGCTGCCATAACCATGAGTG (SEQ ID NO: 44) F3
CTACAGGCATCGTGGTGTC (SEQ ID NO: 45) B3 CTTACGGATGGCATGACAGT (SEQ ID
NO: 46) Loop F TGGGAACCGGAGCTGAAT (SEQ ID NO: 47) Loop B
TCCGATCGTTGTCAGAAGTAAGTTG (SEQ ID NO: 48) Human CFTR FIP
CCAAAGAGTAAAGTCCTTCTCTCTCGAGAGACTGTTGGCCCTTGAAGG (SEQ ID NO: 49)
BIP GTGTTGATGTTATCCACCTTTTGTGGACTAGGAAAACAGATCAATAG (SEQ ID NO: 50)
F3 TAATCCTGGAACTCCGGTGC (SEQ ID NO: 51) B3 TTTATGCCAATTAACATTTTGAC
(SEQ ID NO: 52) Loop F ATCCACAGGGAGGAGCTCT (SEQ ID NO: 53) Loop B
CTCCACCTATAAAATCGGC (SEQ ID NO: 54) Human FIP
GGGCGTGGTAGCGCAGACCAGTCAAGTGATCCTCCTGCCTCAG BRCA-1 (SEQ ID NO: 55)
BIP GAGGTTTCCCTATGTTGCCCAGGCCCAAAGTTCAAGGATCACTTGG (SEQ ID NO: 56)
F3 CAGCCTCAACCTCCTGGGC (SEQ ID NO: 57) B3 TAATCCCAGCATTTTGGGAG (SEQ
ID NO: 58) Loop F GGTCCCAGCTATTTGGAAGG (SEQ ID NO: 59) Loop B
TGGTCTTGAACTTCTGGGC (SEQ ID NO: 60)
[0043] All references cited herein, as well as U.S. application
Ser. No. 13/671,123 filed Nov. 7, 2012 and U.S. Provisional
Application Ser. No. 61/560,518 filed Nov. 16, 2011, are hereby
incorporated by reference.
EXAMPLES
Example 1
Determination of the Difference in Time to Reach Threshold
Amplification Levels for Samples Pre-Incubated at RT Prior to
Amplification with LAMP Primers in the Absence of SSB Compared with
Samples that are Amplified without Pre-Incubation
[0044] LAMP reactions were performed at 65.degree. C. either
immediately or with indicated components incubated for 2 hours at
25.degree. C. Reactions were performed in 25 .mu.L volumes and
consisted of 8U Bst DNA Polymerase (New England Biolabs, Ipswich,
Mass.), 5 ng .lamda. DNA (New England Biolabs, Ipswich, MA), and
LAMP primers used together or separately as shown in FIG. 1A and
1B.
TABLE-US-00002 Primers: (1.6 .mu.M FIP
5'-CAGCCAGCCGCAGCACGTTCGCTCATAGGAGATATGGTAGAGCCGC (SEQ ID NO: 3);
1.6 .mu.M BIP 5'GAGAGAATTTGTACCACCTCCCACCGGGCACATAGCAGTCCTAGGGA
CAGT (SEQ ID NO: 1); 0.2 .mu.M F3 5'-GGCTTGGCTCTGCTAACACGTT (SEQ ID
NO: 4); and 0.2 .mu.M B3 5'-GGACGTTTGTAATGTCCGCTCC. (SEQ ID NO:
5))
[0045] The primers (Integrated DNA Technologies, Coralville, Iowa)
were added to an amplification buffer (20 mM Tris, 10 mM KCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 2 mM MgSO.sub.4, pH 8.8 25.degree. C.)
(New England Biolabs, Ipswich, Mass.) and supplemented with
additional 6 mM MgSO.sub.4, 0.01% Tween-20 and 1.4 mM dNTPs.
[0046] The results are shown in FIGS. 1A-B.
[0047] A lag time of about 15 minutes to reach threshold levels of
amplification was calculated in the sample subjected to 2 hour
pre-incubation before LAMP reached the threshold value. This
decrease in efficiency occurred only when the reaction was
incubated in the presence of primers and DNA polymerase, indicating
unwanted activity of DNA polymerase on primers at RT.
Example 2
Protection of Primers Using SSB from Non-Templated Extension by DNA
Polymerase
[0048] Single LAMP primers with 5'-conjugated fluorophores were
incubated at RT for 2 hours under various conditions to demonstrate
non-template addition by DNA polymerase and inhibition of this
extension by SSB. The primers were incubated at 1.6 .mu.M in
amplification buffer as follows, with all reactions performed in 25
.mu.L volumes: [0049] (i) BIP primer (from set 1 or 2),
amplification buffer, 2 hour incubation at 25.degree. C. no ET SSB.
[0050] (ii) BIP primer, 10U PoID polymerase, amplification buffer,
2 hour incubation at 25.degree. C., no ET SSB. [0051] (iii) BIP
primer, 10U PoID polymerase, amplification buffer, 2 hour
incubation at 25.degree. C., 1 .mu.g ET SSB. [0052] (iv) BIP
primer, 10U PoID polymerase, amplification buffer, 2 hour
incubation at 25.degree. C., 2 .mu.g ET SSB.
[0053] Subsequent to pre-incubation, the primers were diluted to
5nM and analyzed using CE (FIGS. 2A-B). FIGS. 2A-B shows that the
unmodified primer (i) becomes extended in length when a polymerase
is added over the indicated pre-incubation period (ii). This
corresponds to the extension products of the fluorescently-labeled
primer due to DNA polymerase activity. These extension peaks became
diminished in the presence of the increasing amounts of SSB (iii
and iv), indicating inhibition of extension of primer in the
absence of template. FIG. 2A shows data obtained using a 5'-FAM
labeled lambda BIP primer (from primer set 1, see Example 1) and
FIG. 2B shows data obtained using a 5'-FAM labeled lambda BIP
primer (from set 2), demonstrating that non-template primer
extension and SSB protection are not limited to a specific primer
sequence.
Example 3
Determining Optimum Amount of ET SSB for Stimulating Amplification
Rate and Protection from Non-Templated Primer Extension
[0054] LAMP reactions were set up using 1.6 .mu.M FIP and BIP, and
0.2 .mu.M F3 and B3 plus 5 ng .lamda. DNA in a buffer containing 20
mM Tris, 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 2 mM
MgSO.sub.4, pH 8.8 25.degree. C. supplemented with additional 6 mM
MgSO.sub.4, 0.01% Tween-20 and 1.4 mM dNTPs. Reactions were all 25
.mu.L, contained 10U Polymerase D, and were incubated at 65.degree.
C. Threshold time was defined by fluorescence measurement in
Bio-Rad CFX96.TM. (Bio-Rad, Hercules, Calif.) due to presence of 2
.mu.M SYTO-9.RTM. intercalating dye (Life Technologies, Grand
Island, N.Y.). The resulting amplification threshold times are
shown in FIG. 3. The results are shown in FIGS. 3 and 4A-B.
Sequence CWU 1
1
60151DNAArtificial SequenceSynthetic construct 1gagagaattt
gtaccacctc ccaccgggca catagcagtc ctagggacag t 51242DNAArtificial
SequenceSynthetic construct 2caggacgctg tggcattgca gatcataggt
aaagcgccac gc 42346DNAArtificial SequenceSynthetic construct
3cagccagccg cagcacgttc gctcatagga gatatggtag agccgc
46422DNAArtificial SequenceSynthetic construct 4ggacgtttgt
aatgtccgct cc 22522DNAArtificial SequenceSynthetic construct
5ggacgtttgt aatgtccgct cc 22617DNAArtificial SequenceSynthetic
construct 6ctgcatacga cgtgtct 17720DNAArtificial SequenceSynthetic
construct 7accatctatg actgtacgcc 20840DNAArtificial
SequenceSynthetic construct 8aggccaagct gcttgcggta gccggacgct
accagcttct 40920DNAArtificial SequenceSynthetic construct
9aaaactcaaa tcaacaggcg 201020DNAArtificial SequenceSynthetic
construct 10gacggatatc accacgatca 201118DNAArtificial
SequenceSynthetic construct 11gcatcccacc aacgggaa
181211DNAArtificial SequenceSynthetic construct 12cagattaagg a
111342DNAArtificial SequenceSynthetic construct 13cgaactgttt
cgggattgca ttctggaact ccaaccatcg ca 421443DNAArtificial
SequenceSynthetic construct 14ggagcctgca taacggtttc gtcgactcaa
tgctcttacc tgt 431521DNAArtificial SequenceSynthetic construct
15gttggtcact tcgacgtatc g 211620DNAArtificial SequenceSynthetic
construct 16gctcgccgac tcttcacgat 201718DNAArtificial
SequenceSynthetic construct 17tttgcagacc tctctgcc
181820DNAArtificial SequenceSynthetic construct 18ggatttttta
tatctgcaca 201945DNAArtificial SequenceSynthetic construct
19ctgccccgac gataggctta atcgtggtct ggtgaagttc tacgg
452042DNAArtificial SequenceSynthetic construct 20tccagtgcga
cctgctgggt gggtattgtt cgccgccagt ac 422120DNAArtificial
SequenceSynthetic construct 21gatcaccgat ttcaccaacc
202222DNAArtificial SequenceSynthetic construct 22cttttgagat
cagcaacgtc ag 222316DNAArtificial SequenceSynthetic construct
23tgcgccatgt cccgct 162419DNAArtificial SequenceSynthetic construct
24tgagttaacc cacctgacg 192543DNAArtificial SequenceSynthetic
construct 25tgttaaggcg gactgtgttc gtcaaaccgc aacgagacag tct
432643DNAArtificial SequenceSynthetic construct 26ccgagataat
tccaccgttg gatccattcc agcagaacaa gat 432721DNAArtificial
SequenceSynthetic construct 27gatgtcacga aaaattccct c
212819DNAArtificial SequenceSynthetic construct 28gcaatccgag
gatcgtcac 192918DNAArtificial SequenceSynthetic construct
29tgcaaagcac gtggtgcc 183020DNAArtificial SequenceSynthetic
construct 30acacaaactc cagagtgtag 203146DNAArtificial
SequenceSynthetic construct 31ctctgtgaac ggtcatcacc tcgatggctt
gaaccgattg gtatgg 463244DNAArtificial SequenceSynthetic construct
32cttacatggt aatatccagc gtgccacttc accactcgga gcac
443319DNAArtificial SequenceSynthetic construct 33gaacgtctcc
cttcaatcc 193419DNAArtificial SequenceSynthetic construct
34ggaccagaaa tccgtcaca 193520DNAArtificial SequenceSynthetic
construct 35ccgactaccc acatcgttac 203620DNAArtificial
SequenceSynthetic construct 36accttgatgc taaggtggaa
203744DNAArtificial SequenceSynthetic construct 37gattccactt
ccaacgtcgt tgcataggca ttgtatccag agtg 443842DNAArtificial
SequenceSynthetic construct 38cgaagtgaac cttgtcaaca tgagactacc
cacatcgtta cc 423920DNAArtificial SequenceSynthetic construct
39agcaacatag gtttcagttc 204018DNAArtificial SequenceSynthetic
construct 40ctgtgaacgg tcatcacc 184120DNAArtificial
SequenceSynthetic construct 41acggacatgt cgatcatgga
204222DNAArtificial SequenceSynthetic construct 42cgtctccctt
caatccgatg gc 224342DNAArtificial SequenceSynthetic construct
43atgggggatc atgtaactcg cctcgtcgtt tggtatggct tc
424441DNAArtificial SequenceSynthetic construct 44aagcggttag
ctccttcggt ctgctgccat aaccatgagt g 414519DNAArtificial
SequenceSynthetic construct 45ctacaggcat cgtggtgtc
194620DNAArtificial SequenceSynthetic construct 46cttacggatg
gcatgacagt 204718DNAArtificial SequenceSynthetic construct
47tgggaaccgg agctgaat 184825DNAArtificial SequenceSynthetic
construct 48tccgatcgtt gtcagaagta agttg 254948DNAArtificial
SequenceSynthetic construct 49ccaaagagta aagtccttct ctctcgagag
actgttggcc cttgaagg 485047DNAArtificial SequenceSynthetic construct
50gtgttgatgt tatccacctt ttgtggacta ggaaaacaga tcaatag
475120DNAArtificial SequenceSynthetic construct 51taatcctgga
actccggtgc 205223DNAArtificial SequenceSynthetic construct
52tttatgccaa ttaacatttt gac 235319DNAArtificial SequenceSynthetic
construct 53atccacaggg aggagctct 195419DNAArtificial
SequenceSynthetic construct 54ctccacctat aaaatcggc
195543DNAArtificial SequenceSynthetic construct 55gggcgtggta
gcgcagacca gtcaagtgat cctcctgcct cag 435646DNAArtificial
SequenceSynthetic construct 56gaggtttccc tatgttgccc aggcccaaag
ttcaaggatc acttgg 465719DNAArtificial SequenceSynthetic construct
57cagcctcaac ctcctgggc 195820DNAArtificial SequenceSynthetic
construct 58taatcccagc attttgggag 205920DNAArtificial
SequenceSynthetic construct 59ggtcccagct atttggaagg
206019DNAArtificial SequenceSynthetic construct 60tggtcttgaa
cttctgggc 19
* * * * *