Document 5805

Wudpecker Journal of Medical Sciences
Vol. 2(6), pp. 053 - 057, December 2013
ISSN 2315-7240
2013 Wudpecker Journals
Evaluation on the amplification of HPV and EGF genes
by loop-mediated isothermal amplification
Lishi Wang1,2,3, J. H. Maeng#, and E. K. Lee2#*
1
Department of Orthopedics Surgery, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Bioprocessing Research Laboratory, Department of Chemical Engineering, Hanyang University, Ansan, Korea 426-791.
3
Department of Basic Medicine, Inner Mongolia Medical University, Inner Mongolia, 010110, P.R.China
#
College of Bionanotechnology, Gachon University, Seongnam, Korea 461-701
2
#
*Corresponding author E-mail: [email protected] Tel: 82-31-750-8982, Fax: 82-31-750-8769.
Accepted 25 September 2013
Loop-mediated isothermal amplification (LAMP) is a newly developed gene amplification technology. In
this study, we applied the LAMP method to amplification of hEGF (epidermal growth factor) and HPV
(human papilloma virus) genes to compare the result with that from the conventional PCR for efficiency.
We also applied these two methods to a mixture of DNAs. The comparisons showed that LAMP was
simpler and more efficient than the PCR. PCR amplification often has background noises or nonspecific amplification in some cases; while in this aspect, LAMP reaction is more sensitive and specific.
Furthermore, due to its easy manipulation, LAMP technology could allow DNA amplification and gene
detection in small clinics, clinical laboratories, small research laboratories and on-site forensic
applications.
Key words: Isothermal amplification, PCR, rhEGF, HPV66, DNA detection.
INTRODUCTION
Gene amplification is widely used in the upstream
molecular biotechnology, and polymerase chain reaction
(PCR) has been the most popular and widely used gene
amplification method (Yang and Rothman, 2004; Espy et
al., 2006).However, PCR has its own disadvantages; a
laboratory has to be equipped with a thermal cycler and
skillful manipulations which might limit its use. The whole
round requires 2 to 3 hr because of temperature
excursion for denaturation and annealing.
To circumvent this, Notomi et al. (2000) have
developed a new method named loop-mediated
isothermal amplification (LAMP) that can provide 10 9-10 10
times DNA amplification in about 1 hr. This method,
because of its simplicity and specificity, has brought great
benefits to molecular biotechnology applications including
clinical microbiological detections (Seki et al.,2005;
Minami et al., 2006; Cho et al., 2006; Torigoe et al., 2007)
and detection of infectious diseases (Bista et al., 2007;
Toriniwa and Komiya, 2006; Tomlinson et al., 2012).
In this study, we applied the LAMP method to
amplification of hEGF (human epidermal growth factor)
and HPV (human papilloma virus) genes. The result was
compared with that of the conventional PCR method for
efficiency. Furthermore, the LAMP method was applied to
a mixture of DNAs to show its high specificity.EGF gene
PCR product of 171bp was cloned into pGEM-T Vector
(Promega, Madison, WI, USA) and then transformed into
E.coli DH5α pTXB1 (New England Biolabs, USA).
HPV66DNA samples (whole sequence was 7,824bp)
were purified from human serum of an infected patient
(Celltek Co., Ansan, Korea).HPV66 was reported to be
probably the high-risk sexually transmitted HPV that may
be the major cause of invasive cervical cancer (Tawheed
et al., 1991; Clifford et al., 2005). Thus, highly sensitive
and rapid detection of HPV66 is important to detect and
prevent the disease transmission.
For LAMP reaction, we designed two sets of primers for
each of EGF and HPV66 genes (Table1).Amplification
was carried out with a Loopamp DNA amplification kit
(Eiken Chemical Co. Ltd., Japan). PCR was conducted
TM
with Minicycler 3000 (MJ Research Inc., USA).In the
LAMP reaction, the use of 4 primers to recognize 6
distinct regions on the target gene sequence ensured
high specificity of gene amplification. It was reported
when 2 more primers, termed loop primers, were added,
the LAMP reaction time could be shortened to 35 TO 40
min (Nagamine et al., 2002). In some cases, the primer
design is very challenging. For this circumstance, stem
Wang et al.
primers were recently developed (Gandelman et al.,
2011).
RESULTS AND DISCUSSION
Compared to PCR, LAMP method was superior in
simplicity and efficiency in EGF gene amplification. The
use of four primers to recognize six distinct regions on
the target EGF sequence in the initial LAMP steps and
two inner primers (FIP and BIP) in the subsequent steps
ensures the high specificity of EGF gene amplification. In
the case of conventional PCR reaction, an inhibitory
effect might be present during the latter part of cycling,
while LAMP reaction appeared to be limited only by the
amount of deoxynucleotide triphosphates and primers.
In the process of LAMP reaction, a large amount of
pyrophosphate ions was produced, which reacted with
magnesium ions in the reaction mixture to form
magnesium pyrophosphate, a white precipitate byproduct.
Due to its turbidity, the precipitate can be easily
identified with naked eyes, as shown in Figure 1(a).
Following the two amplifications of EGF gene by using
LAMP and conventional PCR, we centrifuged the sample
reaction tube and PCR reaction tube at 4,500rpm for 3
min. The LAMP products tube can be seen clearly with
magnesium pyrophosphate as the by-product precipitate,
while the reaction solution in the conventional PCR tube
was clear with no precipitate.
This turbid precipitate phenomenon allowed easy and
rapid visual detection that the target DNA was amplified
by LAMP, which composes one of the characteristics of
LAMP. The turbidity would be intensified with the LAMP
reaction cycling time and thus the amount of DNA
synthesized. This feature enables the real-time
monitoring of the LAMP reaction by real-time
measurement of turbidity.
In Figure 1(b), we successfully amplified EGF gene by
LAMP method, as shown in Lane 2 and 3, aliquots of 5μL
of EGF LAMP products were loaded in the loading wells
of 1.3% agarose gel (stronger under UV light). The
products appeared as a ladder pattern, with bands of
different sizes from 130bp up to the loading well. Lane 1
is the negative control with no template DNA in the
reaction solution. Lane 4 is the positive control DNA
LAMP fragments. Detailed information about this positive
control DNA is available from the Eiken Company (http://
loopamp.eiken.co.jp).
To confirm EGF LAMP results, the amplified products
were digested with restriction enzyme AluI. We chose this
enzyme because in the restriction sites map of 171bp
EGF gene sequence, there is only one AluI site located at
position 76bp which can split the LAMP polymerized
bands into two fragments of 90bp and 130bp. Thus, if the
amplified EGF products had the exact structures, the
product, after electrophoresed in 1.3% agarose gels
054
would present 90bp and 130bp fragments. Figure 1(c)
shows the exact bands of 90bp and 130bp in Lane 3
according to the NEB DNA Marker.
HPV66gene amplification: Human papillomavirus is a
papillomavirus that infects the epidermis and mucous
membranes of humans. HPV can lead to cancers of the
vagina, vulva, anus, and cervix in women. Over 100
distinct types of HPV have been identified, and
approximately 30 to 40 HPV types infect the anogenital
region through sexual contact. It is reported that type 66
is probably the high-risk sexually transmitted HPV and
may be the major cause of invasive cervical cancer.
Therefore, highly sensitive and rapid detection of HPV is
important to prevent transmission and monitor treatment.
We amplified HPV66 gene by LAMP method. To
confirm the LAMP results, the amplified products were
digested with a restriction enzyme AluI. Figure 1(c)
shows the electrophoretic result of the amplifiedHPV66
by LAMP and PCR. Lane 1 indicates the LAMP product
of HPV66 gene. (Lane 2 is the LAMP product of EGF
gene as a contrast). We could observe that the LAMP
products for HPV66 gene appeared as a ladder pattern.
Lane 3 and 4 indicate LAMP products of HPV66 gene
after digestion with AluI. Restriction enzyme AluI can
divide HPV66 LAMP product into three fragments of
224bp, 188, and 206bp.
These results suggest that HPV66 gene was
successfully amplified by LAMP method. Lane 6 is PCR
product of HPV66 gene. (Lane 5 is PCR product of EGF
gene as a contrast).
To investigate the specificity of LAMP method, we
applied LAMP and PCR method to DNA mixture. We
fixed the LAMP reaction time at 1 hr, since the reaction
was completed in about 1 hr regardless of the starting
concentrations of DNA. LAMP and conventional PCR
were performed with different mixture ratios of HPV66
and pDNA.
As seen in Figure 2(a), HPV66 gene was successfully
amplified by LAMP method in the presence of pDNA. In
the case of conventional PCR, HPV66 gene was
amplified well in the absence of pDNA. However, DNA
amplification was interfered by the presence of unrelated
pDNA (Figure 2b). Therefore, LAMP can be used for
forensic applications, for example, when a gene in a
minute amount of complex specimens needs to be
amplified easily and rapidly.
To compare the yield and purity of the LAMP method,
LAMP and PCR was performed with the same
concentrations (10ng/μL) of EGF and HPV66. The
absorbance ratios of 260/230 (DNA/humic acids) and
260/280 (DNA/protein) were determined. As seen in
Table 1, the yields of amplified EGF and HPV66 gene by
the LAMP method were significantly higher than those by
PCR. Moreover, the absorbance ratios of the LAMP
product were between 1.8 and 2.0, indicating little or no
protein contamination. LAMP, because of its remarkable
characteristics of amplifying nucleic acid with high
055
Wudpecker .J. Med. Sci.
Figure 1. EGF and HPV66gene amplification by LAMP and PCR. (a) Visual detection after centrifugation at 4,500 rpm for 3 min. APCR
products tube with no pyrophosphate precipitate (left tube); a LAMP product tube with pyrophosphate precipitate (right tube); (b)
Electrophoretic image of the LAMP-amplified product, Lane M, NEB 100bp DNA ladder; Lane 1, Negative control (with no template DNA);
Lane 2 and3, EGF gene amplified by LAMP; Lane 4, LAMP product of positive control (template DNA and primer mix were supplied by
Eiken Co. Ltd.); (c) Electrophoretic analysis of the HPV66 LAMP and PCR amplified products. Lane 1, HPV66 LAMP product; Lane 2,
EGF LAMP product; Lane 3 and 4, complete LAMP products of EGF and HPV66 genes after digestion with AluI; Lane 5 and 6, PCR
products of EGF and HPV66 genes.
LAMP reaction:The reaction volume was 25μL by mixing 1.6μM each of FIP and BIP primers, 0.2μM each of F3 and B3 primers, 12.5μL
of 2×Reaction Mix and a certain amount of template DNA (EGF or HPV66). The same reaction mixture without template DNA was used
as a negative control. As a contrast, a positive control reaction was also used.The mixture was heated at 95℃ for 5 min, and cooled on
ice, then 1μL of Bst DNA polymerase was added, followed by incubation at 63℃ for 1 hr and heating at 80℃ for 10 min to terminate the
reaction.
PCR reaction: The total volume was 25 μL with the outer primers F3and B3 as PCR primers, 400μM dNTP, 10×Taqbuffer 2.5μL, a certain
amount of template DNA, and 1u Taq polymerase (New England Biolabs, USA). After the initial denaturation step at 95℃ for 5 min,
thermal cycling at 95℃ for 30s, 62℃ for 30s and 72℃ for 30s was repeated 28 times.Electrophoresis: LAMP and PCR products were
electrophoresed in 1.3% agarose gel followed by staining with ethidium bromide (EB). The gel images were acquired using Chemi-doc
image analyzer (Bio-Rad, Hercules,CA, USA).
Primer sequence sets used to amplify EGF and HPV66 genes:
hEGF: F3 5’-ATGAACTCTGACTCCGAATGC-3’ (171bp)
B35’-CAAGATCTTACGCAGTTCCC-3’
FIP5’-AACACCATCATGCAGGCAATCCGCTGTCTCACGACGGTT-3’
BIP5’-TACATCGGTGAGCGTTGCCAACCATTTCAGGTCGCGATAC-3’
HPV66: F35’- GGT CCA TGA TTA CCT CTG AG -3’ (216bp)
B35’- ATT CCT CCA CAT GGC GAA -3’
FIP5’- ACAAATACCTGATTACCCCAGCATAAACCTTATTGGTTGCAACGT-3’
BIP5’- TGTTGTGGATACTACCAGAAGCACTTGATTTCACGGGCATCA-3’
Figure 2. The specificity test of: (a) LAMP and (b) conventional PCR for the HPV66 gene.
Lane 1 and 5, HPV66 without another DNA; Lane 2 and 6, the mixture ratio of HPV66/pDNA of
1.5/0.5 (w/w); Lane 3 and 7, the mixture ratio of HPV66/pDNA of 1/1 (w/w); Lane 4 and 8, the
mixture ratio of HPV66/pDNA of 0.5/1.5 (w/w).
Wang et al.
056
Table 1. Yield and purity comparison between LAMP and conventional PCR products.
Conc. (ng/μL)
Abs 260/280
Abs 260/230
LAMP
EGF
3,296
1.78
1.88
HPV66
2,912
1.81
1.91
specificity, efficiency and rapidity under isothermal
conditions with a set of four (six) specially designed
primers that recognize six (eight) distinct sequences of a
target DNA, has brought great benefits to applied biosciences.
ADDITIONAL COMMENTS
Primer design and kit components performance
Since the annealing of the four primers to the target DNA
in the LAMP reaction for EGF and HPV66 was critical, we
designed the melting temperature (Tm) of the four primers
0
to be within 58 to 62 C. GC contents were about 50-60%
rich.It was reported that when two more primers, termed
loop primers, were added, the LAMP reaction time could
be shortened to 35 TO 40 min. In that procedure, six
primers recognize eight distinct regions on the target
DNA (Nagamine et al., 2002).
In the LAMP reaction, Bst polymerase is another critical
factor for efficient amplification because of its strand
displacement activity. In the 2×Reaction Mix, the
component betaine (1.6M) has the activity of reducing
base stacking and destabilizing the target DNA helix and
therefore was found to dramatically contribute to the
amplification efficiencies.
We used the negative control as a reaction contrast
because LAMP reaction was very sensitive. On the other
hand, if LAMP reaction was so sensitive that a template
cross-contamination took place, the emergence rate of
the false-positive products would be very high. Therefore,
careful precautions against the template crosscontamination must be taken during sample collections
and preparations for LAMP.
Ever since the development of LAMP method, it has
been used in basic research from a variety of
perspectives. In addition, LAMP is also applied in a wide
range of fields, including SNP typing and quantification of
template DNA. This method is expected to be a more
reliable and more widely used DNA amplification method.
ACKNOWLEDGEMENT
This research was supported by the National Research
Foundation of Korea (NRF) grant funded by the Ministry
of Education, Science and Technology (2008-0061855).
PCR
EGF
264
1.76
1.80
HPV66
471
1.79
1.79
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