© 2000 Oxford University Press Nucleic Acids Research, 2000, Vol. 28, No.

12 e63

Loop-mediated isothermal amplification of DNA

Tsugunori Notomi1,3,*, Hiroto Okayama2, Harumi Masubuchi1, Toshihiro Yonekawa1,
Keiko Watanabe1, Nobuyuki Amino3 and Tetsu Hase1
1EikenChemical Co. Ltd, 1381-3 Shimoishigami, Ohtawara, Tochigi 324-0036, Japan, 2Department of Biochemistry
and Molecular Biology, The University of Tokyo, Graduate School of Medicine, Bunkyo-ku, Tokyo 113-0033, Japan
and 3Department of Laboratory Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871,
Japan

Received February 1, 2000; Revised April 8, 2000; Accepted April 15, 2000

ABSTRACT have been invented. They include nucleic acid sequence-based

amplification (NASBA) (3), self-sustained sequence replication
We have developed a novel method, termed loop-
(3SR) (4) and strand displacement amplification (SDA) (5,6).
mediated isothermal amplification (LAMP), that Each of these amplification methods has its own innovation to
amplifies DNA with high specificity, efficiency and re-initiate new rounds of DNA synthesis. For example, PCR
rapidity under isothermal conditions. This method uses heat denaturation of double-stranded DNA products to
employs a DNA polymerase and a set of four promote the next round of DNA synthesis. 3SR and NASBA
specially designed primers that recognize a total of eliminate heat denaturation by using a set of transcription and
six distinct sequences on the target DNA. An inner reverse transcription reactions to amplify the target sequence.
primer containing sequences of the sense and anti- Similarly, SDA eliminates the heat denaturation step in cycling
sense strands of the target DNA initiates LAMP. The DNA synthesis by employing a set of restriction enzyme digestions
following strand displacement DNA synthesis and strand displacement DNA synthesis with modified nucleo-
primed by an outer primer releases a single-stranded tides as substrate.
DNA. This serves as template for DNA synthesis These methods can amplify target nucleic acids to a similar
magnitude, all with a detection limit of less than 10 copies and
primed by the second inner and outer primers that
within an hour or so, but still have shortcomings to overcome
hybridize to the other end of the target, which (7,8). They require either a precision instrument for amplification
produces a stem–loop DNA structure. In subsequent or an elaborate method for detection of the amplified products
LAMP cycling one inner primer hybridizes to the loop due to poor specificity of target sequence selection. Despite the
on the product and initiates displacement DNA simplicity and the obtainable magnitude of amplification, the
synthesis, yielding the original stem–loop DNA and a requirement for a high precision thermal cycler in PCR
new stem–loop DNA with a stem twice as long. The prevents this powerful method from being widely used, such as
cycling reaction continues with accumulation of 109 in private clinics as a routine diagnostic tool. On the other
copies of target in less than an hour. The final products hand, NASBA and 3SR, which do not use thermal cycling, are
are stem–loop DNAs with several inverted repeats of compromised in specificity, resulting mainly from the necessity to
the target and cauliflower-like structures with multiple use a relatively low temperature of 40°C for amplification.
SDA largely overcomes these shortcomings by using four
loops formed by annealing between alternately
primers and isothermal conditions for amplification, but still
inverted repeats of the target in the same strand.
has weak points: increased backgrounds due to digestion of
Because LAMP recognizes the target by six distinct irrelevant DNA contained in the sample and the necessity to
sequences initially and by four distinct sequences use costly modified nucleotides as substrate. Although the use
afterwards, it is expected to amplify the target of multiple primers, such as in nested PCR and SDA, has
sequence with high selectivity. improved amplification specificity for the target sequence,
residual co-amplification of irrelevant sequences still causes a
general setback in nucleic acid amplification, particularly for
INTRODUCTION
diagnostic use.
Nucleic acid amplification is one of the most valuable tools in We have recently developed a novel method that can amplify
virtually all life science fields, including application-oriented a few copies of DNA to 109 in less than an hour under
fields such as clinical medicine, in which diagnosis of infectious isothermal conditions and with greater specificity. We describe
diseases, genetic disorders and genetic traits is particularly the mechanism, sensitivity and specificity of this amplification
benefited by this new technique. In addition to the widely used method, termed loop-mediated isothermal amplification
PCR-based detection (1,2), several amplification methods (LAMP).

*To whom correspondence should be addressed at: Eiken Chemical Co. Ltd, 1381-3 Shimoishigami, Ohtawara, Tochigi 324-0036, Japan. Tel: +81 287 29 2000;
Fax: +81 287 29 3456; Email: [email protected]
e63 Nucleic Acids Research, 2000, Vol. 28, No. 12

MATERIALS AND METHODS and 1 mM EDTA, followed by neutralization with 1 M Tris–HCl

(pH 8.0), and staining with SYBR Green I.
DNA oligonucleotides
Amplification of HBV DNA cloned in pBR322
Primer BIP for M13mp18 (M13BIP) consisted of the comple-
mentary sequence (24 nt) of B1, a TTTT linker and B2 (24 nt): HBV viral DNA (type adr) was cut with BamHI, inserted into
5′-CGACTCTAGAGGATCCCCGGGTAC-TTTT-TGTTGT- the BamHI site of pBR322, digested with EcoRV and then
GTGGAATTGTGAGCGGAT-3′. Primer FIP for M13mp18 used as template. The LAMP reaction mixture was the same as
(M13FIP) consisted of F1c (25 nt), a TTTT linker and the com- for M13mp18 DNA except for the use of 1.6 µM each
plementary sequence of F2c (22 nt): 5′-ACAACGTCGT- HBVFIP and HBVBIP primers and 0.2 µM each HBVF3 and
GACTGGGAAAACCCT-TTTT-GTGCGGGCCTCTTCGC- HBVB3 primers. The LAMP reaction was carried out at 60°C
TATTAC-3′. Primers B3 and F3 for M13mp18 (M13B3 and for 45 min. Aliquots of 2 µl of the amplification products were
M13F3) were 5′-ACTTTATGCTTCCGGCTCGTA-3′ and 5′- mixed with 300 µl of 1/10 000 diluted original SYBR Green I
GTTGGGAAGGGCGATCG-3′, respectively. The probes in 10 mM Tris–HCl (pH 8.0), and 1 mM EDTA, incubated at
used for Southern blot hybridization were 5′-AAGCTTGGC- room temperature for 30 min and quantified for fluorescent
ACTGGCCGTCGT-3′ (M13-281) and 5′-GTTACCCAACT- intensity with a Shimadzu RF-5000 spectrophotometer.
TAATCGCCTTGCAGCACAT-3′ (M13-333). The primers
used for amplification of the HBs region of hepatitis virus B RESULTS AND DISCUSSION
(HBV) DNA were 5′-GATAAAACGCCGCAGACACATC-
CTTCCAACCTCTTGTCCTCCAA-3′ (HBVBIP), 5′-CCTG- The LAMP method
CTGCTATGCCTCATCTTCTTTGACAAACGGGCAACAT-
This method relies on auto-cycling strand displacement DNA
ACCTT-3′ (HBVFIP), 5′-CAAAATTCGCAGTCCCCAAC-3′
synthesis that is performed by a DNA polymerase with high
(HBVB3) and 5′-GGTGGTTGATGTTCCTGGA-3′
strand displacement activity and a set of two specially
(HBVF3). The primers used for prostate-specific antigen
designed inner and two outer primers. In the initial steps of the
mRNA amplification were 5′-TGTTCCTGATGCAGT-
LAMP reaction, all four primers are used, but later during the
GGGCAGCTTTAGTCTGCGGCGGTGTTCTG-3′ (PSABIP),
cycling reaction only the inner primers are used for strand
5′-TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAA-
displacement DNA synthesis. The inner primers are called the
TACCTGGCCTG-3′ (PSAFIP), 5′-TGCTTGTGGCCT-
forward inner primer (FIP) and the backward inner primer
CTCGTG-3′ (PSAB3) and 5′-GGGTGTGGGAAGCTGTG-3′
(BIP), respectively, and each contains two distinct sequences
(PSAF3).
corresponding to the sense and antisense sequences of the
Reaction mixture for LAMP target DNA, one for priming in the first stage and the other for
self-priming in later stages. For ease of explanation, the
LAMP was carried out in a total 25 µl reaction mixture sequences (typically 23–24 nt) inside both ends of the target
containing 0.8 µM each FIP and BIP, 0.2 µM each F3 and B3, region for amplification in a DNA are designated F2c and B2,
400 µM each dNTP, 1 M betaine (Sigma), 20 mM Tris–HCl respectively (Fig. 1). Two inner sequences (typically 23–24 nt)
(pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 4 mM MgSO4, 0.1% 40 nt from the ends of F2c and B2 are designated F1c and B1
Triton X-100 and the specified amounts of double-stranded and two sequences (17–21 nt) outside the ends of F2c and B2
target DNA. The mixture was heated at 95°C for 5 min, then are designated F3c and B3. Given this structure, the sequences
chilled on ice, 8 U Bst DNA polymerase large fragment (New of FIP and BIP were designed as follows. FIP contains F1c, a
England Biolabs) were added, followed by incubation at 65°C TTTT spacer and the sequence (F2) complementary to F2c.
for 1 h and heating at 80°C for 10 min to terminate the reaction. BIP contains the sequence (B1c) complementary to B1, a
TTTT spacer and B2. The two outer primers consist of B3 and
Analysis of product
the sequence (F3) complementary to F3c, respectively. A DNA
Aliquots of 5 µl of LAMP products and 1 µl of the products sample containing the target sequence and the four primers is
digested with restriction enzymes were electrophoresed in 2% heat denatured and rapidly cooled on ice. The LAMP reaction
agarose gels (0.5× TBE) followed by staining with SYBR is then initiated by addition of the Bst DNA polymerase large
Green I (Molecular Probes Inc.). Southern blot analyses were fragment and carried out at 65°C for 1 h.
performed by transfer to Hybond N+ nylon membrane The mechanism and expected reaction steps of LAMP are
(Amersham-Pharmacia). Oligonucleotide probes end-labeled illustrated in Figure 1. Inner primer FIP hybridizes to F2c in
with a DIG Oligonucleotide Tailing Kit (Roche Diagnostics) the target DNA and initiates complementary strand synthesis
were used for detection according to the manufacturer’s (Fig. 1A). Outer primer F3, which is a few bases shorter and
protocol. An aliquot of 5 µl of LAMP products was also run for lower in concentration than FIP, slowly hybridizes to F3c in
14 h in a 0.7% alkaline agarose gel containing 50 mM NaOH the target DNA and initiates strand displacement DNA

Figure 1. (Next page) Schematic representation of the mechanism of LAMP. (A) Steps in the LAMP reaction. This figure shows the process that starts from primer
FIP. However, it should be remembered that DNA synthesis can also begin from primer BIP. (B) Schematic presentation of the structure of LAMP products in a
linearized DNA form. B+, B–, F+ and F– stand for the DNA structures shown in the boxes on the left. +, the target sequence flanked by B1 and F1c; –, the
complementary sequence.

ii
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e63 Nucleic Acids Research, 2000, Vol. 28, No. 12

synthesis, releasing a FIP-linked complementary strand, which

can form a looped out structure at one end (structure 4). This
single-stranded DNA serves as template for BIP-initiated DNA
synthesis and subsequent B3-primed strand displacement
DNA synthesis, leading to the production of a dumb-bell form
DNA (structure 6), which is quickly converted to a stem–loop
DNA by self-primed DNA synthesis (structure 7). This stem–
loop DNA then serves as the starting material for LAMP
cycling, the second stage of the LAMP reaction.
To initiate LAMP cycling, FIP hybridizes to the loop in the
stem–loop DNA (structure 7) and primes strand displacement
DNA synthesis, generating as an intermediate one gapped
stem–loop DNA with an additional inverted copy of the target
sequence in the stem and a loop formed at the opposite end via
the BIP sequence (structure 8). Subsequent self-primed strand
displacement DNA synthesis yields one complementary structure
of the original stem–loop DNA (structure 10) and one gap
repaired stem–loop DNA with a stem elongated to twice as Figure 2. (A) Nucleotide sequence of M13mp18 used for designing the inner
long (double copies of the target sequence) and a loop at the and outer primers. The nucleotide sequence of the sense strand of M13mp18
opposite end (structure 9). Both these products then serve as DNA is shown. DNA sequences used for primer design are shown by heavy
template for a BIP-primed strand displacement reaction in the lines. Probe sequences used for Southern blot hybridization are indicated by
dotted lines and the restriction sites for BamHI, PstI and PvuII are indicated by
subsequent cycles, a part of which is designated the elongation
boxes. Numbers at the left end correspond to the positions in M13mp18 (GenBank
and recycling step, illustrated in the right half of Figure 1A. accession no. X02513). (B) Schematic representation of the anticipated structure
Thus, in LAMP the target sequence is amplified 3-fold every of the amplified product. B+, B–, F+ and F– in the first row are as in Figure 1B.
half cycle. The second row indicates the probes used for Southern blot hybridization. The
The final products are a mixture of stem–loop DNAs with restriction sites for PstI, PvuII and BamHI are shown as lines and the sizes of
the restriction fragments are in the boxes.
various stem lengths and cauliflower-like structures with
multiple loops formed by annealing between alternately
inverted repeats of the target sequence in the same strand
(Fig. 1, structures 16–18). The structures of the cycling inter- To confirm the structure, the amplified products were
mediate and final products are schematically illustrated in digested with several restriction endonucleases and their sizes
Figure 1B in linearized DNA form.
analyzed by electrophoresis. BamHI cuts B1, PstI cuts between
The use of four primers (recognition of six distinct
F1c and B1, and PvuII cuts between F1c and F2c (Fig. 2A).
sequences) in the initial steps of LAMP and two primers
(recognition of four distinct sequences) during the subsequent Consequently, if the amplification products had exactly the
steps ensures high specificity for target amplification. Moreover, structures shown in Figure 1, the products would be frag-
in LAMP four primers (six distinct recognition sequences) are mented to 101 and 230 bp fragments by BamHI digestion,
simultaneously used to initiate DNA synthesis from the original 137 and 194 bp fragments by PstI digestion, and 237, 315 and
unamplified DNA to generate a stem–loop DNA for subsequent 347 bp fragments by PvuII digestion. As shown in Figure 3A,
LAMP cycling, during which the target is recognized by four the sizes of the fragments generated were approximately
sequences. Therefore, target selectivity is expected to be 100 and 230 bp for BamHI, 140 and 200 bp for PstI, and 240,
higher than those obtained in PCR and SDA. 320 and 350 bp for PvuII digestion (Fig. 3A, lanes 5–7), in
good agreement with the predicted sizes (Fig. 2B). To further
LAMP amplification of M13 DNA as a model confirm their structure, the restriction digests were analyzed by
In order to demonstrate the mechanism, the efficiency, the Southern blot hybridization using the sequence complementary to
specificity and the ease of use of LAMP, we chose M13mp18 the inner region between F1c and B1 (M13-281), the sequence
DNA as a model target DNA and prepared four primers that complementary to the inner region between F1c and F2c
met the LAMP requirements (Fig. 2A). The reaction was (M13-333), and the BIP primer itself as hybridization probes.
carried out at 65°C for 1 h and the products were separated by M13-281 and M13-333 hybridized to the 194 bp PstI product,
agarose gel electrophoresis and identified by restriction but not to the 137 bp fragment (Fig. 3B and C, lane 6). In
enzyme digestion and Southern blot hybridization with appro-
contrast, BIP hybridized to the 137 bp, but not the 194 bp
priate probes (Fig. 2A and B). The LAMP reaction produced
fragment (Fig. 3D, lane 6). Similarly, Southern blot results of
many bands of different sizes from ~300 bp to the loading well
(Fig. 3A, lane 4). Production of the bands depended on the the PvuII and BamHI digests perfectly agreed with the conclusion
presence of the inner primers, the template and DNA that the amplified DNA originated from target M13 DNA
polymerase. When the products were analyzed by alkaline (Fig. 3B–D, lanes 5 and 7). The structures of the amplified
agarose gel electrophoresis, smeared DNA between bands and products were also confirmed by cloning and sequencing.
at the well (shown in Fig. 3A, lane 4) was shifted to bands of Several bands shown in Figure 3A, lane 4, were isolated and
<10 kb (Fig. 3E). Thus, we attributed the slow migrating DNA cloned after digestion with mung bean nuclease. The
and the DNA in the loading well to replicating intermediates sequences of the cloned DNAs perfectly agreed with the
containing single-stranded loops, as shown in Figure 1A. expected nucleotide sequences (data not shown).
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Figure 3. Restriction analysis and Southern blot hybridization of the amplified M13mp18 DNA. (A) Electrophoretic analysis of the LAMP amplified M13mp18
product. Six hundred copies of M13mp18 DNA were amplified by LAMP with the specific primers designed on the sequences shown in Figure 2 and run on a 2%
agarose gel followed by SYBR Green I staining. Lane M, 100 bp ladder used as size marker (New England Biolabs); lane 1, M13mpl8 DNA digested with PvuII;
lane 2, LAMP without Bst DNA polymerase; lane 3, LAMP without target M13 DNA; lane 4, complete LAMP; lanes 5–7, complete LAMP products after digestion
with BamHI, PstI and PvuII, respectively (one fifth of the digests were loaded). (B–D) Southern blot analysis of the LAMP products. The 2% agarose gel shown
in (A) was used for Southern blot hybridization with M13-281 DNA (B), M13-333 DNA (C) and M13BIP (D) as probes. (E) Alkaline agarose gel electrophoresis
of the LAMP products. Lane m, λ DNA HindIII digests; lane 4, the same sample as in (A).

Optimized conditions for LAMP which reduce base stacking (9–11), stimulated not only the
overall rate of the reaction, but also increased target selectivity
Since hybridization of the four primers to the target DNA in
with a significant reduction in amplification of irrelevant
the initial step was critical for the efficiency of LAMP, the
sequences.
sequences and sizes of the primers were chosen so that their
melting temperatures (Tm) fell within certain ranges. The F2 Sensitivity of LAMP
and B2 sequences in FIP and BIP were chosen such that their
Tm values fell between 60 and 65°C, the optimal temperature LAMP is highly sensitive and able to detect DNA at as few as
for Bst polymerase. The Tm values of F1c and B1c were set six copies in the reaction mixture. As shown in Figure 4A, in a
45 min LAMP reaction six copies of the HBV target were
slightly higher than those of F2 and B2 in order that a looped
amplified to a detectable level. As expected, use of inner
out structure formed immediately after release of the single-
primers that do not form a looped out structure led to no ampli-
stranded DNA from the template. Furthermore, the Tm values
fication (Fig. 4B, lanes 5–8). In the absence of one of the outer
of the outer primers (F3 and B3) were set lower than those of
primers no significant amplification occurred with 60 copies of
F2 and B2 in order to ensure that synthesis occurred earlier
the HBV target (Fig. 4B, lanes 2–4), indicating a strict require-
from the inner primers than from the outer primers. In addition,
ment for recognition of six distinct sequences in the target
the outer primers were used at 1/4–1/10 the concentration of DNA in LAMP.
the inner primers.
LAMP seems less prone to the presence of irrelevant DNA
The formation of a stem–loop DNA (sees Fig. 1, structure 7) than PCR. The presence of 100 ng of human genomic DNA in
from a dumb-bell structure (structure 6) is critical for LAMP the LAMP reaction for six copies of HBV target neither signifi-
cycling. We examined the effect of various sizes of loop cantly adversely affected the amplification efficiency nor
between F2c (B2c) and F1c (B1c) on amplification efficiencies generated significant background (Fig. 4C). Single PCR,
and found that a loop of 40 bases or longer gave the best results which was performed under the same conditions as for LAMP,
(data not shown). failed to amplify six copies of HBV in the absence and
The efficiency of LAMP depends on the size of the target 60 copies in the presence of 10–100 ng of the genomic DNA,
DNA because one rate limiting step for amplification in this though nested PCR overcame this problem (Fig. 4D).
method is strand displacement DNA synthesis. We tested
various sizes of target DNA and found that the best results LAMP for a RNA target
could be obtained with 130 to 200 bp DNAs. DNA of more LAMP is also applicable to RNA upon use of reverse tran-
than 500 bp amplified, but very poorly. Therefore, the size of scriptase (RTase) together with DNA polymerase (12). This
target DNA should be set to less than 300 bp, including F2 and method (reverse transcription-coupled LAMP) easily detected
B2. prostate-specific antigen (PSA) mRNA in one PSA-expressing
DNA polymerase is another critical factor for efficient LNCaP cell mixed with 1 000 000 PSA-negative K562 cells
amplification. The best amplification was obtained with Bst (13–15; Fig. 5, lane 6). This amplification depended on both
polymerase or BcaBEST DNA polymerase (TaKaRa) for less RTase and Bst polymerase (Fig. 5, lanes 2 and 4) and the
than 10–23 mole target DNA. Z-Taq DNA polymerase product was authenticated by Sau3AI digestion (Fig. 5, lanes 8
(TaKaRa) was less efficient under the current conditions, but and 9).
might be useful when polymerase has to be added before heat
denaturation of the target DNA, because it is thermostable. Advantages of LAMP
Chemicals destabilizing the DNA helix were found to markedly (i) LAMP amplifies DNA with high efficiency under
elevate amplification efficiencies in LAMP. The presence of isothermal conditions without a significant influence of the co-
0.5–1.5 M betaine (N,N,N-trimethylglycine) or L-proline, presence of non-target DNA. Its detection limit is a few copies,
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Figure 4. Sensitivity of LAMP. (A) Time course of the LAMP reaction with various amounts of HBV DNA. Various numbers of copies of HBV DNA were amplified
by LAMP. At various times, the reaction was terminated and the amounts of products quantified by measuring fluorescence intensity of SYBR Green I. (B) Requirements
for primers in the LAMP reaction. Sixty copies of HBV DNA were amplified by LAMP with omission of one or two of the primers. The products were electro-
phoresed in 2% agarose gels and stained. –, the corresponding primer was omitted from the reaction; B2 and F2, BIP and FIP were replaced by B2 and F2, respectively,
which do not contain B1c and F1c and, therefore, are unable to form the looped out structure. (C) The effect of the presence of genomic DNA on sensitivity. Various
numbers of copies of HBV DNA were amplified at 60°C for 60 min in the absence or presence of 100 ng of human genomic DNA and the products separated by
gel electrophoresis. Lane M, 100 bp ladder size markers (TaKaRa); lanes 1–4, LAMP carried out in the absence of human genomic DNA; lanes 5–8, LAMP carried
out in the presence of 100 ng genomic DNA; lanes 1 and 5, LAMP without HBV DNA; lanes 2 and 6, with six copies; lanes 3 and 7, with 60 copies; lanes 4 and 8,
with 600 copies of HBV DNA. Lanes 9 and 10 are EarI digests (1/5 vol) of the same amplified DNAs as in lanes 2 and 6, respectively. (D) Nested PCR of HBV
DNA under similar conditions. Single and nested PCR reactions were performed in a 50 µl reaction mixture containing 2.5 U AmpliTaq Gold (PE Biosystems),
0.2 µM each primer (first PCR, HBVB2/HBVF2 or HBVB1/HBVF1; second PCR, HBVB1/HBVF1), 1× GeneAmp PCR buffer (10 mM Tris–HCl, pH 8.3, 50 mM
KCl, 1.5 mM MgCl2, 0.01% gelatin) and 0.2 mM dNTPs. The sequences of the primers used were 5′-CCAACCTCTTGTCCTCCAA-3′ for HBVB2, 5′-GACAA-
ACGGGCAACATACCTT-3′ for HBVF2, 5′-GGATGTGTCTGCGGCGTTTTATC-3′ for HBVB1 and 5′-AGAAGATGAGGCATAGCAGCAGG-3′ for HBVF1.
Both the first and second round nested PCRs were carried out as follows: preincubation at 95°C for 10 min; 40 cycles each of 30 s at 95°C, 30 s at 60°C and 1 min
at 72°C. One microliter of the first PCR products (HBVB2/HBVF2) was subjected to second PCR. An aliquot of 10 µl of the reaction products was analyzed by
4% agarose gel (0.5× TBE) electrophoresis followed by staining with SYBR Green I.

being comparable to that of PCR. (ii) The products are a

mixture of stem–loop DNAs with various sizes of stem and
cauliflower-like structures with multiple loops induced by
annealing between alternately inverted repeats of the target
sequence in the same strand, the latter of which would enable
their simple, easy and selective detection, such as via
mechanisms similar to multivalent antigen–antibody inter-
actions. (iii) LAMP is highly specific for the target sequence.
This is attributable to recognition of the target sequence by six
independent sequences in the initial stage and by four
independent sequences during the later stages of the LAMP
reaction. This partly alleviates the general problem of back-
Figure 5. Detection of PSA mRNA by reverse transcription-coupled LAMP grounds associated with all nucleic acid amplification
(RT-LAMP). Various numbers of LNCaP cells were mixed with 106 PSA-non-
producing K562 cells and total RNA was extracted. RT-LAMP was carried out methods. (iv) LAMP is simple and easy to perform once the
in the same reaction mixture as for M13mp18 DNA amplification except that appropriate primers are prepared, requiring only four primers,
1.6 µM each PSAFIP and PSABIP, 0.2 µM each PSAF3 and PSAB3, 0.8 M a DNA polymerase and a regular laboratory water bath or heat
betaine, 5 mM DTT, 16 U Bst polymerase, 100 U ReverTra Ace (Toyobo) and
5 µg of extracted RNA were used. All the above components were mixed at block for reaction. (v) By combination with reverse transcription,
once on ice and were incubated at 65°C for 45 min. The products were electro- LAMP can amplify RNA sequences with high efficiency.
phoresed in 2% agarose gel followed by SYBR Green I staining. + and –, RT-
LAMP carried out in the presence and absence of Bst DNA polymerase or
ReverTra Ace, respectively. Lanes 8 and 9, the same products (1/5 vol) as in ACKNOWLEDGEMENT
lanes 6 and 7, respectively, but digested with Sau3AI; lane M, 100 bp ladder
(New England Biolabs). We thank Dr K. Tatsumi for helpful comments on this manuscript.
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