Advanced primer design

40

41

6. Designing wild type and mutant type primers
In PrimerExplorer, it is possible to introduce mutations into the target sequence and then design primers. However, if
there are too many mutations, the primer design conditions become too stringent and either the primers are not generated
or the variety is insufficient. In such a case, one can design the primer with less stringent condition, for example reduce
the number of the mutation point entered or completely eliminate the mutation sites from the target sequence. Appropriate
primer sets could be selected, 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 and initiates the genesynthesis. If the mutation is at the 3’ end of F2 (B2), the DNA polymerase has difficulty in recognizing 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 the both wild type and mutant typeare 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 thetarget region for amplification.
Figure 6.2 shows an example of primer selection. Under default, primers have been designed for the wild type strain.
Here, we focus on the 25 primer set candidates that contain the mutations and select the common primers. A star shows
the locations of the mutations, and the locations of the mutations applicable to the primers designed are enclosed by adotted line. This can be used to confirm the location of the primers for a given mutation. Table6-2 shows these results. The
mark (plus, +) indicates the location of the primer region (5’ end, internal region, 3’ end) for each primer set (F3, F2, F1, B1,
B2, B3) for a given mutation. It can be determined that No 1, No 6 - 11, and No 16 - 25 are the primers less susceptible tothe effects of the mutations at amplification. These are selected from the primer list, and their detailed information is thenassessed for a final selection of the primer sets.

6.2 Highly specific primers (specific primers that distinguish between wild-type and mutant 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 can select highly specific primers. If
the primers include a mutation in this location, the mutant type is generally amplified while the wild type amplification ishindered, thus improving the specificity toward the mutant type (table 2.1).

3442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), or c). In Table 22, 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 specificF31)Common primer4)

2)

5’

In

●

●

primers
3)

3’

F2

5’

In

●

●

●

Specific primer5)

F1c
3’

●

5’

B1c

In

3’

●

●

●

5’

B2

In

3’

5’

In

●

●

●

●

B3
3’

●

5’

In

●

●

●

1) 5’ terminal region2) internal region3) 3’ terminal region4) Common primer : Mutation sites that can be permitted for the amplification of wild type and mutant type using common primers.
5) Specific primer : Sites for mutation for distinguishing between wild type and mutant type.
6) ＋ : The mutation points are included.

**

M13_3.nucM13_3M1.nuc

1:GCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG 60
1:GCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTGCAACGTCGTGACTGGGATAACCCTG 60

M13_3.nucM13_3M1.nuc61:GCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCG 120
61:GCGTTACCCAACTTAATCGACTTGCAGCACATCCCGCTTTCGCCAGCTGGCGTAATAGCG 120

*

*

*

*

M13_3.nuc
121:AAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCT 180
M13_3M1.nuc 121:AAGAGTCCCGCACCGATCGCCCTTCCCAACACTTGCGCAGCCTGAATGGCGAATGGCGCT 180*

M13_3.nuc
181:TTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTG 240
M13_3M1.nuc 181:TTGCCTGGTTTCCGGCACCAGAAGCGGTGCAGGAAAGCTGGCTGGAGTGCGATCTTCCTG 240
M13_3.nuc
241:AGGCCGATACGGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCT 300
M13_3M1.nuc 241:AGGCCGATACGGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCT 300
M13_3.nuc
301: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

35
43

510
510

3’

●

*

*

*

*

*

*

Figure 6.2 Primer sets and location of mutations

36
44

*

No.

F3
5’

In.

F2
3’

5’

＋
＋
＋

2
3
4

＋

5

＋
＋
＋
＋

6
7
8
9

14
15

＋
＋
＋

18

＋
＋
＋
＋
＋

In.

B2
3’

5’

In.

B3
3’

5’

In.

Primer
3’

＋
＋
＋
＋
＋

＋
＋
＋
＋

＋
＋
＋
＋
＋
＋
＋

20

＋
＋

22

＋
＋
＋

Common*Specific**SpecificSpecificSpecific

CommonCommon＋＋＋＋＋CommonSpecificSpecificSpecificSpecific
CommonCommon＋Common

＋
＋

19

25

5’

Common

＋
＋
＋
＋
＋
＋
＋
＋
＋

13

17

B1c
3’

Common

12

16

In.

Common

11

24

5’

＋

10

23

F1c
3’

＋

1

21

In

CommonCommon＋＋＋＋＋CommonCommonCommonCommonCommonTable 6.2 Primer sets depending on location of mutation*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 type3745

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 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 the mutation 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 indicatedin Figure 7.2, the indicator of the location of the mutation changes from a star (*) to a hyphen (-). This status indicates thatthe 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 window

38
46

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). Here, the primers will be designed based on there-entered mutation information (See Figure 7.3). 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
1,000 primer sets have been designed (See Figure 7.4).

39
47

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

The mutation is
located here.

Primer sets are generated
Figure7.5 Primer Set List window (page 1)

40
48

that avoids this location.

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’ and 3’ ends of each primer region.
For example, if a mutation is introduced into a particular region of the target sequence as described in the preceding
section, the number of primer set candidates for amplifying the gene containing this region decreases dramatically. Whenamplifying the region that contains the mutation, it is preferred that the mutation be not located within the primer; however,there are some cases in which such strict conditions dramatically reduce the number of potential primers, or even noprimers are produced. If no mutations are present in the region, on the other hand, many primer set candidates
incorporating the location of the mutation are designed, as shown in Figure 7.6. Hence, a wide variety of primer
candidates are generated by using less stringent conditions to allow the mutation to be present within the primer region.
The primers with the mutation having the minimal influence on the amplification are then chosen.
In PrimerExplorer, it is possible to select primer regions that contain the mutation. Regions that can be selected are the
5’ and 3’ ends, as well as the internal region for regions F3, B3, F2, B2, F1c, and B1c. Since the 5’ ends of regions F3, B3,
F2, and B2, the 3’ ends of regions F1c and B1c, and the internal regions between the 5’ and 3’ ends do not serve as thestarting points of the amplification, they are less susceptible to the influence of the mutation. If primer design can hardly beachieved, allow mutations at these regions to proceed to the primer design.
First, the primers are designed so that the mutation is located at the 5’ end of the primer region. As shown in Figure 7.7,
check the box for the 5’ end at region F3 in the Primer Design window, and then click on the “Generate” button to designprimers that include the mutation at the 5’ end.

41
49

Use the mutation
information

2) Click on the“Generate”
button.

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

1) Check the “F3 5’term” box.

Figure7.7 Primer design window

4250

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 have specified 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, the process ofprimer 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’ end
The locationof region F3.

containing themutation is shownin 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 and then click on
the “Generate” button. After the primers have been designed, click on the “Display” button to display the data.
Primers with the mutations at the F3 5’ end or the F2 5’ end are designed. (See Figure 7.10)

4351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

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

4452

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 and mutation sites
are displayed with reference to the genes at the top sequence. The alignment should be performed with the Genetyx orClustal 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
and click on the “Primer Design” button, and you will find that, as seen in the figure below, the window displays consensus
sequences (*) and mutation sites (-) based on SeqA. Using three results, you can design primers (Figure 8.2).

Figure 8.1 Multiple alignment1) Click on the“Common”
button.

2) Click on the“Generate”
button.
Figure 8.2 Multiple alignment read window

53
45

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, these primers are relatively insusceptible to the mutations. Figure
8.4 shows the details of the primer sets.
Mutations are recognized

Mutations are recognized at

at the 5’ end and internal

the 5’ end and internal region

region of the F3 primer.

of the F2 region.

Figure 8.3 Primer Set List window

46
54

Figure 8.4 Primer Set Details window

55
47

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 toautomatically design specific primer sets.

1) Click on the2)
Figure9.1 Primer design window

Figure9.2 Primer Set List window

56
48

Click

on

the

“Generate” button.

“Specific” button.

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

Figure 9.3 Primer Set Details window

57
49

9.2 Design in Expert Mode

Click on the“Generate” button.

Allow themutations at the
5’end of F1c/B1cor at the 3’end of
F2/B2.

Figure9.4 Primer design window

5850

As shown in Figure 9.4, Expert Mode enables designingwith allowance for mutations to be included at the ends ofeach primer.
The results in Expert Mode are shown in Figure 9.5. Primer
sets are generated that can recognize mutations sites atthe 3’ end of 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 the target genes. Primers are designed for theentire target region. If too many primer sets have been
generated, you should set stricter design conditions andlimit the number of primer sets to be generated. You cando this as instructed in p17-27, Section 1, to select thedesired number of primer sets.

Figure 9.5 Primer Set List window

51
59

Experimental procedure and techniquesof LAMP method

60

61

Experimental procedures and techniques

In conducting the LAMP method, the greatest
care is needed to prevent contamination.
One preventive measure is to keep separate thepreparation of reaction solutions, addition of
template, and detection. The experimental
equipment and experimental locations should be
kept
physicallyseparate.
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 be donein 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,butforfurther
assessments,
HPLC-grade purity should be used.)6252

A basic procedure for confirmation of LAMP productis restriction enzyme digest and electrophoresis.
The Figure on the left is the electrophoresis result of
the LAMP products obtained using M13 as template.
From the left are the size marker, 600 molecules, 60
molecules, 6 molecules of the template and negative
control.
There exists a single HindIII site in the target M13
sequence. In the figure on the right, from the left are:size markers, two lanes of untreated sample, and asample digested with HindIII. The apparent digestion
result by HindIII treatment has confirmed theamplification 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 thereaction, resulting in a high risk of contamination.
Thus, we recommend that electrophoresis is
only for the initial confirmation of amplification
and afterwards detection can be conductedwithin the tube. Examples include a fluorescentreal-time detection or real time turbidity
detection.
The figure on the left shows the results obtainedwith real time fluorescent detection with M13 as the-17
template. The amount of template is stated as 10-23
mol/tube to 10
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 degree of
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 such as
the template or primer is reconstituted in water, thedeterioration may be accelerated, thus they should
be stored in a buffer such as TE. Specifically, thetarget template DNA at low concentration can easily
be degraded and thussufficient care is needed.

63
53

List of terms

64

65

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 a low
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←
Secondline
andbeyondisthedata
tggcttcaaccttagctggggctgcagcagcac>AA987701(genbank-upd) ← Multiple sequences are entered in thisformat 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 the left and the 3’ end
on the right, the 5’ → 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)6654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

Locus name, sequence length, molecule type, GenBank division, and modification date
Brief description of sequence
Original accession number

SOURCE

Key words to describe the entry for searches
Organism from which DNA is derived

ORGANISM

Formal scientific name for the source organism

REFERENCE
COMMENT

Literature reference
Biological function or database information

FEATURES

Information about genes and gene products, as well as regions in the sequencesourcemisc_signal
mRNA
CDS
intronMutation
BASE COUNT
ORIGINA 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
51 atatactcac//

aatctctggt ttattgtgca
agcataactg tatatacacc

gtttatggtt
cagggggcgg

ccaaaatcgc
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

55
67

around 65°C.
The two important features of isothermal amplification are: ① DNA synthesis occurs by DNA
polymerase with strand displacement activity without the need for thermal cycles fordouble-strand denaturation ⇒ annealing - ⇒ DNA synthesis ① 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 F2

F3Primer 5’ F3 3’
3’
5’

3’

F3c F2c F1c Target DNA
F3

B1

B2 B3

B1c B2c B3c

F2 F1

3’

BIP

B3

5’
3
’

5’ B3 Primer

3’
B2
B1c 5’

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

F2c
(2)

F1
c
F1 3’

B1
5’

B2
B1c

(3)

56
68

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 × 1,000/(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 (2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acid, a chelator which
can remove 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, elevationin temperature leads to destruction of the hydrogen bonds responsible for the base pairing. The
temperature at which the DNA loses the double stranded structure, so that 50% is double
stranded structure and 50% is single stranded, is the Tm. There are three hydrogen bonds in
the GC pair and two in the AT pair, so that DNA with higher content of GC base pairs is resistantto 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 substances, it can result in the movement of substances to thepositive or negative electrode. In applying the current, the buffer, filters, gels, carrier ampholytes,
are used.
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In nucleic acid electrophoresis, relatively large molecular weight DNA (60 - 100k bp) are
separated by agarose gels, while smaller molecular weight DNA (1 k bp 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 becomes unable tobind 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 sequence ofa gene can result in a change in the genetic makeup. Gene mutation can be at the level of the
genome, chromosome, a part of a chromosome, gene or nucleotide. Depending on how a genehas 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.

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Version5 / date : August 201959