Advanced primer design_6. Designing wild type and mutant type primers
In PrimerExplorer V4, it is possible to introduce mutations into the target sequence and then designprimers.

However, if there are too many mutations, the primer design conditions become too stringent andeither the primers are not generated or the variety is insufficient. In such a case, one can design theprimer with less stringent condition, for example reduce the number of the mutation point entered orcompletely eliminate the mutation sites from the target sequence.

Appropriate primer sets could beselected, while identifying where the mutation points in the target sequence are located relative to theprimer.

6.1 Detecting wild type and mutant type by amplification using common primers
In general, the primers are design to exclude mutation within the primer region, but if there are numerous
mutations, it may not be possible to design primers that satisfy these conditions.

For this reason, primers

are designed that allow (contain) mutations and if possible, try to design primers that are not likely
influenced by the mutation.
Under the principles of the LAMP reaction, F2 of FIP (or B2 of BIP) anneals to the target gene andinitiates the gene synthesis. If the mutation is at the 3’ end of F2 (B2), the DNA polymerase has difficulty inrecognizing the double strand formed between the primer and the target gene, thus inhibit the gene
amplification. Similar principles apply to the 5’ end of F1c (B1c) and the 3’ end of F3 (B3). Therefore,
primers are selected so that mutations are not located in these regions.
On the other hand, if primers are selected so that the mutations are outside of the 3’ end in F2 (B2), 5’
end of F1c (B1c), or 3’ end of F3 (B3), the primers are less susceptible to the effect of the mutation and theboth wild type and mutant type are detectable by a common set of primers.
Thus, primers are selected by permitting the mutation to be located within the following locations (Table
6-1).
a) 3’ end of F1c or B1c and in the internal regionb) 5’ end of F2 or B2 and in the internal regionc) 5’ end of F3 or B3 and in the internal region
Here, we will design common primers that detect M13 and its mutant. Figure 6-1 shows an alignment of
the wild type and the mutant type. In the entire length of 510 bp there are seven mutations. The region
containing these mutations is the target region for amplification.
Figure 6-2 shows an example of primer selection. Under default, primers have been designed for thewild type strain.

Here, we focus on the 25 primer set candidates that contain the mutations and select thecommon primers.

A star shows the locations of the mutations, and the locations of the mutations

applicable to the primers designed are enclosed by a dotted line.
of the primers for a given mutation.

This can be used to confirm the location

Table6-2 shows these results. The mark (plus, +) indicates thelocation of the primer region (5’ end, internal region, 3’ end) for each primer set (F3, F2, F1, B1, B2, B3) fora given mutation. It can be determined that No 1, No 6 - 11, and No 16 - 25 are the primers lesssusceptible to the effects of the mutations at amplification. These are selected from the primer list, andtheir detailed information is then assessed for a final selection of the primer sets.

35

6.2 Highly specific primers (specific primers that distinguish between wild-type andmutant type)
If the mutant type and wild type need to be distinguished, a method opposite to the aforementioned
method is used.

Thus, by selecting primers with the mutation in the locations mentioned below, one canselect highly specific primers. If the primers include a mutation in this location, the mutant type is generally
amplified while the wild type amplification is hindered, thus improving the specificity toward the mutant type(table 2- 1).
a) 5’ end of F1c or B1cb) 3’ end of F2 or B2c) 3’ end of F3 or B3
As in (1), from the primer list designed under default, primer sets are selected that fulfill the above a), b), orc). In Table 2- 2, No 2 – 5 and No 12 - 15 are mutant type-specific primer sets. Then, their detailed
information is examined for final selection of the primer set.

Table 6-1 Select Common primers and specific primers
Primer region

F3

Position

a)

In

)

＋

5’

Common primer
Specific primer

d)

e)

＋ｆ

b)

F2
c)

3’

＋

5’

In

＋

＋

F1c
3’

＋

5’

B1c

In

3’

＋

＋

＋

5’

B2

In

3’

5’

In

＋

＋

＋

＋

B3
3’

＋

＋

5’

In

＋

＋

3’

＋

a) 5’ terminal region, b) internal region, c) 3’ terminal region, d) Common primer : Mutation sites that can be permitted for theamplification of wild type and mutant type using common primers. e) Specific primer : Sites for mutation for distinguishing
between wild type and mutant type. f) ＋ : The mutation points are included.

M13_3.nucM13_3M1.nuc**
1: GCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG
60
1: GCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTGCAACGTCGTGACTGGGATAACCCTG 60

M13_3.nucM13_3M1.nuc**61: GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCG
120
61: GCGTTACCCAACTTAATCGACTTGCAGCACATCCCGCTTTCGCCAGCTGGCGTAATAGCG 120**
M13_3.nuc121: AAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCT180
M13_3M1.nuc 121: AAGAGTCCCGCACCGATCGCCCTTCCCAACACTTGCGCAGCCTGAATGGCGAATGGCGCT 180*
M13_3.nuc181: TTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTG
240
M13_3M1.nuc 181: TTGCCTGGTTTCCGGCACCAGAAGCGGTGCAGGAAAGCTGGCTGGAGTGCGATCTTCCTG 240
M13_3.nuc241: AGGCCGATACGGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCT 300
M13_3M1.nuc 241: AGGCCGATACGGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCT 300
M13_3.nuc301: ACACCAACGTAACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGA 360
M13_3M1.nuc 301: ACACCAACGTAACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGA 360
M13_3.nuc361: CGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGC 420
M13_3M1.nuc 361: CGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGC 420
M13_3.nuc421: GAATTATTTTTGATGGCGTTCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAA 480
M13_3M1.nuc 421: GAATTATTTTTGATGGCGTTCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAA 480
M13_3.nuc481: CGCGAATTTTAACAAAATATTAACGTTTAC
M13_3M1.nuc 481: CGCGAATTTTAACAAAATATTAACGTTTAC

Figure 6-1 Alignment of wild type with mutant type

36

510
510

＊

＊

＊

＊

Figure 6.2 Primer sets and location of mutations＊＊

Figure 6.2 continued

37＊

Table 6-2 Primer sets depending on location of mutation
F3

No.
5ʼ

In.

F2
3ʼ

5ʼ

＋
＋
＋

2
3
4

＋

5

＋
＋
＋
＋

6
7
8
9

14
15

＋
＋
＋

18

5ʼ

＋
＋
＋
＋
＋

In.

B2
3ʼ

5ʼ

In.

B3
3ʼ

5ʼ

In.

＋
＋
＋
＋
＋

＋
＋
＋
＋

＋

20

＋
＋

22

＋
＋
＋

Common*Specific**SpecificSpecificSpecific

＋
＋
＋
＋
＋
＋

CommonCommon＋＋＋＋＋CommonSpecificSpecificSpecificSpecific
CommonCommon＋Common＋＋19

Primer3ʼ

Common

＋
＋
＋
＋
＋
＋
＋
＋
＋

13

17

B1c
3ʼ

Common

12

16

In.

Common

11

24

5ʼ

＋

10

23

F1c
3ʼ

＋

1

21

In

CommonCommon＋＋＋＋＋CommonCommonCommonCommon25*Common : primer set candidates that can amplify both wild type and mutant type with a common primer set**Specific : primer set that distinguishes mutant type from wild type38

Common7. Primer design that takes the location of mutation into account
7.1 Uploading the target sequenceThis section explains primer design for amplifying the wild type and mutant type strains together using
common primers, or for selectively amplifying the mutant type only.
Open the PrimerExplorer V4 startup window, use the steps outlined in Section 1 to select the target
sequence file (see p. 13) and then click on the “primer design button.” (Figure not shown)

7.2 Designing primers that do not include the mutation by entering the location of themutation over the target sequence
We now explain the design of primers that do not include the location of the mutation. Click on the“Mutation” button after specifying the location of the mutation in the target sequence in the primer design
window (Figure 7.1). As indicated in Figure 7.2, the indicator of the location of the mutation changes from astar (*) to a hyphen (-). This status indicates that the entry has been accepted.

To delete this mutation

information, click on the “Clear” button.

2) Click on the“Mutation”
button.

1) Specify thelocation of themutation in thesequence.

Figure7.1 Primer design window39

To delete themutation
information, clickon the “Clear”
button.

The star (*)
changes to ahyphen (-).

Figure7.2 Window after entering the location of mutation

Now, we will enter a mutation at another location (a different mutation).
designed based on the re-entered mutation information (See Figure 7.3).

Here, the primers will be

The primers are designed to

avoid including the mutation.

This mutation
information will
be used.

Click on the“Generate”
button.

Figure7.3 Window after entering site of mutation
1000 primer sets have been designed (See Figure 7.4).

40

1,000 primer sets
have been designed.

Click on the“Display”button toshow the result.
Figure7.4 Primer design window

Next, click on the “Display” button to show the results. As indicated in Figure 7.5, the primers aredesigned to exclude mutation from the primer regions.

<Note>The process of primer design when a mutation has been entered is to determine first theprimer location for each of F1, F2, F3, B1, B2, and B3, then delete those candidates that include
to the location of the mutation, and then use the remaining primer regions to compose theprimer sets.

The mutation is
located here.

Primer sets are generated
that avoids this location.

Figure7.5 Primer Set List window (page 1)

41

For reference, Figure 7.6 shows the results obtained when the primer design was conducted without any
mutation entry.

Figure7.6 Primer set designed with no mutation entry

7.3 Designing primers with mutations at the 5’ end or 3’ end of the primer region
Here we discuss designing primers with mutations located at the 5’ end and 3’ end of the primer region.
For example, in the example discussed above, if a mutation is located near 141 bp in the target sequence,then the number of primer set candidates for amplifying this region decreases dramatically.

When

amplifying the region that contains the mutation, it is preferred that the mutation not be located within theprimer, but there are circumstances where such strict conditions greatly decreases the number of potential
primers or prohibits primers from being designed at all. If the mutation is not present in the primer, then as
indicated in Figure 6.6, many primer set candidates are generated such that they incorporate the location of
the mutation.

Thus, by using less stringent conditions to design the primers to allow the mutation to bepresent within the primer regions, many varieties of primer candidates can be generated. Then, primers
are chosen such that the mutation has the least influence on the amplification.
In PrimerExplorer V3 it is possible to select the primer regions that contain the mutation. Regions thatcan be selected are 5’ end, 3’ end and the internal region for the F3, B3, F2, B2, F1c, and B1c. Since the 5’
end at regions F3, B3, F2, and B2 and the 3’ end of regions F1c and B1c and the internal regions between
5’ end and 3’ end do not serve as the starting points of the amplification and are less susceptible to theeffect of the mutation. When primer design can hardly be achieved, allow mutations at these regions toproceed to the primer design.
First, the primers are designed so that the mutation is located at the 5’ end of the primer region. As in
Figure 6.7, check the box for the 5’ end at region F3 in the primer design window, and then click on the“Generate” button to design primers that include the mutation at the 5’ end.

42

Use the same
mutation
information as in
Figure 7.3.

2) Click on the“Generate”
button.

3) After the primers havebeen designed, click on the“Display” button.

1) Check the“F3 5’ term”
box.

Figure7.7 Primer design window

43After the primers have been designed, click on the “Display” button to display the data.

As indicated in

the Figure 6.8 Primer Set List window, the mutation in the primer is indicated in red.

Since we havespecified that the mutation be present at the 5’ end of the primer at regionF3, we will design primers that
include the mutation at the 5’ end of region F3.

<Note>
In case mutation regions have been specified using “Mutation/ Consensus” setting, theprocess of primer design will not eliminate primer regions that contain the specified mutation
regions (for example F3 5’ end) among the generated candidate primer by its filter. These
primers incorporate with other remaining primer candidates to compose primer sets.

Primer is designedwith the mutation
located at the 5’ endof region F3

The location
containing themutation isshown in red.

Figure7.8 Primer Set List (page 1)

It is possible to select multiple regions that contain the mutation.

Here, mutations are allowed at the 5’

ends of locations F3 and F2.
As indicated in Figure 7.9, check the box for F3 5’ end and the F2 5’ end in the primer design window andthen click on the “Generate” button.

After the primers have been designed, click on the “Display” button todisplay the data.
Primers with the mutations at the F3 5’ end or the F2 5’ end are designed. (See Figure 7.10)

441) Check the“F2 5’ term”
box.

2) Check the“F3 5’ term”
box.

Figure7.9 Primer design window

Primers with amutation at the 5’ endof region F3 or F2 aredesigned.

Figure7.10 Primer Set List window (page 1)

45

Next, we design primers that contain a mutation at the 3’ end of a primer region.

As in Figure 7.11, in

the primer design window, check the box at the 3’ end of F2 region and click on the “Generate” button.
After designing the primers, click on the “Display” button to display the results.
When a mutation is present at the 3’ end of regions F2, F3, B2, or B3 or at the 5’ end of regions F1c andB1c, the specificity of the primer to the wild type strain becomes low, and the specificity toward the mutant
type strain becomes high. These locations serve as the replication starting points after annealing, so thatthe gene with the mutation is specifically amplified. Using this property, one can choose highly specificprimers.

1) Check the“F2 3’ term”
box.

Figure7.11 Primer design window

46

Figure 7.12 shows the result of primer designing that includes the mutation at the 3’ end of F2 region.

Mutation is
located at the 3’
end of F2.

Figure7.12 Primer Set List window (page 1)

The method described in Section 1 is then followed (See p.22 - 23) to compare the primer informationand select the primer sets.

47

8. Designing common primer using multiple alignment results
8.1 Reading multiple alignment results
If alignment results are input in the same way for normal gene sequences, consensus sequences andmutation sites are displayed with reference to the genes at the top sequence. The alignment should be
performed with the Genetyx or Clustal W program. Here, take an alignment of the three genes SeqA, SeqB,
and SeqC for an example. Figure 8.1 shows an example alignment of SeqA, SeqB, and SeqC that was
performed with Genetyx. Read the results in PrimerExplorer Ver.4 and click on the “Primer Design” button,
and you will find that, as seen in the figure below, the window displays consensus sequences (*) andmutation sites (-) based on SeqA. Using three results, you can design primers (Figure 8.2).

Figure 8.1 Multiple alignment
Normal1 Alignment.txt1) Click on the“Common”
button.

2) Click on the“Generate”
button.

Figure 8.2 Multiple alignment read window48

8.2 Designing common primers
Check the “Common” button and press the “Generate” button, and you will find that five common primers
are generally designed. As shown in Figure 8.3, sets of these primers are generated with mutations
included at the 5’ end and internal region of F3, F2, B3, or B2, or at the 3’ end and internal region of F1c or
B1c. Since the mutations are recognized at locations other than the starting points of amplification, theseprimers are relatively insusceptible to the mutations. Figure 8.4 shows the details of the primer sets.

Mutations arerecognized at the
5’ end and internal
region of the F3
primer.

Mutations arerecognized at the
5’ end and internal
region of the F2
region.

Figure 8.3 Primer Set List window

49

Figure 8.4 Primer Set Details window50

9. Specific primer design
9.1 Design in Easy Mode
As shown in Figure 9.1, check the “Specific” button at the bottom right of the window and press the“Generate” button to automatically design specific primer sets.
Normal1 Alignment.txt2) Click on the“Generate”
button.

Figure9.1 Primer design window

Figure9.2 Primer Set List window

511) Click on the“Specific”
button.

As shown in the primer design result window in Figure 9.2, primer sets are generated that can recognize
mutations sites at the 3’ end of F3/B3 or F2/B2, or the 5’ end of F1c/B1c. Figure 9.3 shows the details of theprimer sets.

Figure 9.3 Primer Set Details window52

9.2 Design in Expert Mode

Click on the“Generate”
button.

Allow the mutations at
the 5’ end of F1c/B1c orat the 3’ end of F2/B2

Figure9.4 Primer design window53

As shown in Figure 9.4, Expert Modeenables designing with allowance formutations to be included at the ends ofeach primer.
The results in Expert Mode are shown in
Figure 9.5. Primer sets are generated thatcan recognize mutations sites at the 3’ endof F3/B3 or F2/B2, or at the 5’ end of
F1c/B1c. Specific primers are generated
from the 5’ end toward the 3’ end of thetarget genes. Primers are designed for theentire target region. If too many primer sets
have been generated, you should set
stricter design conditions and limit the
number of primer sets to be generated.
You can do this as instructed in p19-23,
Section 1, to select the desired number ofprimer sets.

Figure 9.5 Primer Set List window

54

Experimental procedure and techniquesof LAMP method_Experimental procedures and techniques

In conducting the LAMP method, the greatest
care is needed to prevent contamination.
One preventive measure is to keep separate
the preparation of reaction solutions, addition of
template, and detection.
The experimental
equipment and experimental locations should be
kept physically separate.
If separately
experimental locations are not available, at least
conduct the preparation of reaction solutions andthe addition of templates in separate clean
benches. However, the detection should bedone in a separate room.
Handling of the amplification product might cause
contamination, thus sufficient care is needed
when performing electrophoresis or restriction
enzyme digests for the purpose of identifying theamplification products.

In the LAMP method, the amplification conditionsof Loopamp DNA Amplification Kit are regarded
as the fundamental experiment conditions.
To increase the amplification speed or sensitivity,
consider the following factors.
・ Amount of enzyme, amount of primer(particularly the Inner primer)・ Reaction temperature (In addition to therecommended temperature of 63°C, temperaturein the 60 - 65°C range can also be considered)・ Purity of the Inner primer (for screening theprimer sets, the de-salting grade purity is
sufficient, but for further assessments, HPLCgrade purity should be used.)55

A basic procedure for confirmation of LAMP
product is restriction enzyme digest andelectrophoresis.
The Figure on the left is the electrophoresis resultof the LAMP products obtained using M13 as
template. From the left are the size marker, 600
molecules, 60 molecules, 6 molecules of thetemplate and negative control.
There exists a single HindIII site in the target M13
sequence. In the figure on the right, from the leftare: size markers, two lanes of untreated sample,
and a sample digested with HindIII. The apparent
digestion result by HindIII treatment has confirmed
the amplification of the target template.

A basic procedure for confirmation of LAMP isby electrophoresis, but this requires that the tube
lid be opened after completion of the reaction,
resulting in a high risk of contamination. Thus, werecommend that electrophoresis is only for the initialconfirmation of amplification and afterwards
detection can be conducted within the tube.
Examples include a fluorescent real-time detection
or real time turbidity detection.
The figure on the left shows the results obtainedwith real time fluorescent detection with M13 as the
template. The amount of template is stated as 10-17
mol/tube to 10 - 23 mol/tube, and the speed ofamplification relates to the quantity of the template.
The figure on the right is real time turbidity
detection with λ DNA as the template. Samples
NC1 to NC4 are negative controls, and PC1 to
PC4 are positive controls, indicating a high degreeof reproducibility.

There may be circumstances where the LAMP
amplification proceeds quite effectively in the
beginning, but as time passes it begins to perform
poorly. In such circumstances, the deterioration of
the reagents might be a possible cause, therefore,
reagents should be handled with sufficient care.
Generally the precautions required are similar to
those for PCR, storing the reagents at -20°C, andstoring the undiluted primer stock solution at -80°C.
The substrate dNTP also deteriorates gradually andthus needs to be handled with care. If DNA suchas the template or primer is reconstituted in water,
the deterioration may be accelerated, thus theyshould be stored in a buffer such as TE.
Specifically, the target template DNA at low
concentration can easily be degraded and thus
sufficient care is needed.

56

List of terms

_List of terms
AT rich, GC rich:
The GC content of nucleic acids can vary for different creatures, or it can differ depending onwhether the nucleic acid is derived from prokaryotic cell or non-prokaryotic cell. Those with alow GC content is said to be AT rich, and those with a high GC content is said to be GC rich.
bp:
Abbreviation for base pairs. Every nucleic base can specifically combine with its counterpart
nucleic base through hydrogen bonds. This plays an important role in nucleic acid replication,transcription and interaction between mRNA and tRNA. In DNA the pairing is between adenine(A) and thymine (T) or between guanine (G) and cytosine (C), while in RNA the pairing is
between A and uracil (U), or between G and C. The length of double-stranded DNA is often
expressed as the number of bases (bp).
dNTP:
A solution containing equal amounts of dATP, dTTP (or dUTP), dGTP and dCTP, and is usedas substrate in nucleic acid synthesis. During nucleic acid synthesis, if the dNTP concentration
is too high in the reaction solution, it is said that the mismatch of nucleotide will increase.
FASTA format:
FASTA is a computer program that, through database searches, identifies similarities in geneor protein sequences. It is appropriate for searching through long sequences for homologies.
The FASTA format is a format most often used in sequence analysis programs and is in thefollowing format.
>AA987701(genbank-upd) ← The header is a comment beginning with > (sequence name or origin)taaagaagtaagcctttatttccttgttttgca ← Second line and beyond is the data
tggcttcaaccttagctggggctgcagcagcac>AA987701(genbank-upd) ← Multiple sequences are entered in this format
taaagaagtaagcctttatttccttgttttgca
tggcttcaaccttagctggggctgcagcagcac

Forward side, backward side:
At the start of DNA synthesis, primers are needed; PCR requires the minimum of two, while in
the LAMP method the minimum of four primers are needed.
With respect to the
double-stranded DNA, with the coding region of the relevant gene as shown in the 5’ end on theleft and the 3’ end on the right, the 5’ Æ 3’ direction is the forward side and the reverse is the
backward side.
For PCR
Forward Primer
5’
3’
3’
5’

5’
3’
5’
3’
Backward Primer(Reverse Primer)

For the LAMP method
p.47 the LAMP method to legend (1)57GC content：
In expressing the base composition of nucleic acids, the proportion (percent) of G and C in theentire sequence. The GC content of primers is selected so that that it is not AT rich to ensurestability of binding to the target gene. For duplex nucleic acids, the base pairing is fixed, so thisindicates the proportion of (G+C) in the entire sequence. The GC content is one index of aproperty of nucleic acids. The DNA GC content differs depending on the organism and in
higher animals is in a narrow range centered at 42%, while in bacteria, it ranges from 75-25%.
GenBank format：
GenBank is an internationally known public DNA database maintained by the US NCBI
(National Center for Biotechnology Information). GenBank uses the database entry format
indicated below.
LOCUS
DEFINITIONACCESSIONKEYWORDS
SOURCEORGANISM
REFERENCE
COMMENTFEATURES
sourcemisc_signal

mRNA
CDS
intronMutation
BASE COUNT
ORIGIN//

Locus name, sequence length, molecule type, GenBank division, and modification date
Brief description of sequence
Original accession number
Key words to describe the entry for searches
Organism from which DNA is derived
Formal scientific name for the source organism
Literature reference
Biological function or database information
Information about genes and gene products, as well as regions in the sequence
A region of sequence, source organism
A region of sequence, function or signal typeA region of sequence, mRNAA region of sequence, protein coding region
A region of sequence, intron location
Sequence change due to mutation within the sequence
The number of A, C, G, T, or other letter in a sequence
Letter corresponding to sequence start

1 gaattcgata aatctctggt ttattgtgca gtttatggtt ccaaaatcgc
51 atatactcac agcataactg tatatacacc cagggggcgg aatgaaagcg
Symbol indicating sequence end

Hind III:
Type of restriction enzyme often used in experimental procedures. This enzyme is produced
from Haemophilus influenzae Rd and thus the enzyme is so named. The recognition sequenceand location of the cuts are indicated below.
5’……A AGCTT……3’
3’……TTCCA A……5’
HPLC purification:
A purity grade for synthetic oligonucleotides.
The LAMP method:
The LAMP method (Loop-mediated isothermal Amplification) is a simple, rapid, specific andcost-effective nucleic acid amplification method solely developed by Eiken Chemical Co., Ltd.
As a gene amplification technology, compared to PCR, it has higher specificity and amplification
efficiency, and has the advantage that it can perform the amplification at a constant temperature
58

around 65°C.
The two important features of isothermal amplification are: c DNA synthesis occurs by DNA
polymerase with strand displacement activity without the need for thermal cycles fordouble-strand denaturation ⇒annealing - ⇒DNA synthesis d Four primers (recognizing 6
distinct regions in the target gene) are used to amplify the gene, whereby the loops formed at theend of the synthesized strand proceed to self-primed DNA synthesis. The following Figure
describes the steps in amplification.
(1)

FIP

5’ F1c F23’F3
F3Primer 5’
3’F3c F2c F1c Target DNA
3’5’
F3 F2 F1B1 B2 B3B1c B2c B3c
3’

BIP

3’
B2

B3

5’
3’

5’ B3 Primer

B1c 5’

Through DNA polymerase with strand displacement activity, the following
reactions proceed at a constant temperature of about 65°C.

B1

F1c
(2)

F2c
F1 3’

5’

B2
B1c

(3)

59

By adding four primers (1), after a few steps of reaction, single stranded structure with a loop
structure at each end is formed. (2) This serves as the starting point where primers will anneal toproceed to the amplification. As a result of this process, various sized structures consisting ofalternately inverted repeats of the target sequence on the same strand are formed.
Loopamp DNA Amplification kit:
A reagent kit designed for LAMP amplification for research use. The contents are: the buffer,
substrate and DNA polymerase with strand displacement activity. The user prepares the LAMP
primers designed for the target gene of interest. The system can then be used in a variety of
applications.
Loop primers:
The Loop Primers (either Loop Primer B or Loop Primer F), containing sequencescomplementary to the single stranded loop region (either between the B1 and B2 regions, or
between the F1 and F2 regions) on the 5' end of the dumbbell-like structure, provide anincreased number of starting points for DNA synthesis by the LAMP method. Loop primers
provide an increased number of starting points for DNA synthesis resulting in shorter
amplification time and higher specificity.
M13 phage:
Filamentous single-stranded DNA phage. Infects the host E coli through the F pili, and isincorporated into the bacteria. In the host, the single-stranded DNA changes into adouble-stranded replicative form, and this template is used to form single-stranded DNA. Oncetaken up by the newly formed progeny phage particles, the host bacteria do not lyse but rather
releases phage particles. This phage is also useful as a cloning vector, and is widely used toprepare single stranded DNA for use in DNA sequencing by the dideoxy method.
Nearest-Neighbor method:A method for estimating the Tm of DNA and has now come to be the standard. The Tm isdetermined by taking into account the thermodynamic factors of the neighboring bases under
the following formula.
Tm = ΔH × 1000/(ΔS+R ln(C/4)) - 273.15 + 16.6 log[Na+]R: gas constant = 1.987cal/ºC/mol
ΔH: enthalpy (kcal/mol)
ΔS: entropy (eu)
C: oligonucleotide concentration (M)[Na+]: sodium ion concentration

PCR:
PCR (polymerase chain reaction) involves repeated DNA synthesis reactions of a specified
DNA sequence bracketed by 2 primers and can amplify the specified DNA region by several
10,000 folds. The amplification primers used are usually the synthetic oligonucleotides
containing the bases at both ends of the region to be amplified, and the reaction involves
repeated steps of 1) denaturation of double-stranded DNA, 2) annealing of oligonucleotide, and
3) DNA polymerase-mediated complementary strand synthesis (repeated usually 20-30 times).
Developed by Cetus Co., in 1985.
TE buffer：
Nucleic acid buffer (10mM Tris- HCl (pH 8.0), 1 mM EDTA). Contains divalent metal ion(Mg2+ etc) chelator EDTA (ethylenediamine- N,N,N’,N’- Tetraacetic acid, a chelator which canremove divalent metal ions in samples), which inhibits the divalent metal-ion dependent
nucleases (nucleic acid degrading enzyme) and preserves the nucleic acids.
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Tm:
Refers to the melting temperature of biological polymers. For nucleic acid in solution,
elevation in temperature leads to destruction of the hydrogen bonds responsible for the basepairing. The temperature at which the DNA loses the double stranded structure, so that 50% isdouble stranded structure and 50% is single stranded, is the Tm. There are three hydrogenbonds in the GC pair and two in the AT pair, so that DNA with higher content of GC base pairs is
resistant to thermal denaturation and thus has higher Tm. The efficiency of binding to primers
is generally expressed as Tm.
Annealing:
After the double-stranded DNA has been denatured to the single-stranded form, annealingrefers to the re-binding of the denatured single-stranded DNA back into a double-stranded DNA.
The DNA-specific double stranded helix is formed, so that the annealing can be called
renaturation. With double-stranded DNA, heat or alkaline treatment causes denaturation into
single-stranded DNA. Denatured double stranded DNA under certain conditions forms thehydrogen bonds and forms a complete double helical structure.
Oligo concentration:
In the text, the oligo concentration in the slide on p43 refers to the oligonucleotide
concentration, namely the primer concentration.
5’ end, 3’ end:
In nucleic acids all nucleotides are bound to the next nucleotide via phosphate diesterification
between the 5-carbon on the 5-carbon sugar to the 3-carbon on the next sugar. At both ends
these exist as -OH groups, called the 5’ end and 3’ end. For a given nucleic acid, the left handside is generally the 5’ end or upstream, while the right hand side is the 3’ end or downstream.
Cloning:
Gene cloning refers to the isolation of DNA in which unfractionated DNA fragments areinserted into vectors to form recombinants, which are introduced and propagated in the hosts ascolonies or plaques and then the desired DNA is identified, isolated and purified.
Free energy:
A type of thermodynamic coefficient. This refers to the thermodynamic equilibrium standard
under standard experimental conditions. In systems where change of state is possible, thechange tends to go in the direction of lowest free energy. As with chemical reactions, in thechemical equilibrium state, the free energy of the system is minimized. Currently used freeenergy terms include the Gibbs free energy and the Helmholtz free energy.
Restriction enzyme:
Name of enzyme that recognizes and cuts DNA at specific sequences. Classified as types I,
II and III depending on the needed cofactors for enzyme activity and the type of cut made.
Widely distributed among microorganisms, the enzyme type or recognition sequence differs
depending on the microbial strain, so that numerous types are available.
Real-time turbidity detection:
Gene amplification is conducted by LAMP and its detection can be done by simultaneously
monitoring the white turbidity caused by the existence of magnesium pyrophosphate, theamplification by-products. Thus, real time detection can be achieved. The detection of themagnesium pyrophosphate turbidity is facilitated by the efficiency and specificity of the LAMP
amplification reaction.
Electrophoresis:
Method for separation and analysis of DNA. It takes advantage of the observation that whenelectric current applied to a substances, it can result in the movement of substances to thepositive or negative electrode. In applying the current, the buffer, filters, gels, carrierampholytes, are used.

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In nucleic acid electrophoresis, relatively large molecular weight DNA (60 - 100kbp) are
separated by agarose gels, while smaller molecular weight DNA (1 kbp or less) are separatedusing acrylamide gels.
Secondary structure:
The primer secondary structure refers to the hairpin structure that can form if the primer iscomplementary to itself. Depending on the primer sequences, the likelihood for hairpin
formation can differ greatly. If the primer itself forms hairpin structures, the primer becomesunable to bind to the target gene or can bind to unexpected genes, thus resulting in false
positives.
Primer:In general, the term primer refers to the oligonucleotide that forms a double strand with thetarget gene and supplies the 3’-OH needed to initiate the DNA polymerase-mediated elongation
reaction. Through the activity of the DNA polymerase, the complementary nucleotides areadded to the 3’ –OH group of the primer on the template DNA sequence, so that the elongation
proceeds from the 5’ side to the 3’ side.
Primer dimer:This term refers to the structure that forms when a primer hybridizes to another primer. In thegene amplification method involving DNA synthesis in a test tube, it is necessary to have theprimer concentration in the reaction mix at a concentration that is far greater than the Target
gene concentration, so that if the structure of the primer permits the hybridization of a primer toitself, primer dimers can form and thus inhibit the hybridization to the Target gene.
Plain text format:
Containing only the sequences information in the following format.
ctcgaggact ggggaccctg caccgaacat ggagaacaca acatcaggat tcctaggacc
cctgctcgtg ttacaggcgg ggtttttctt gttgacaaga atcctcacaa taccacagag
tctagactcg tggtggactt ctctcaattt tctaggggga gcacccacgt gtcctggccc
Mutation:
Refers to spontaneously occurring gene mutation. Changes occurring in the base sequenceof a gene can result in a change in the genetic makeup. Gene mutation can be at the level ofthe genome, chromosome, a part of a chromosome, gene or nucleotide. Depending on how agene has changed, the mutation can be classified also as a point mutation, deletion, duplication,
inversion, insertion, translocation, etc. Mutations can occur in various forms, so its effect at thelevel of expression can vary greatly from noticeable significant changes to those only detectable
after statistical analysis.
Terminal end stability:
The stability of the formation (ease of formation) of double stranded region formed between
the template gene and each primer at the 3’ end and the 5’ end. The LAMP Primer designing
software determines the stability as the ΔG (change in free energy) calculated by the NearestNeighbor method.

62_Version date: January 2009