Question

Discuss the primers used in LAMP (FIP, BIP, F3, B3, LF and LB). How are they designed?

What are their roles in each stage of the reaction? What should the range of Tm's be for each of

them? What relative concentrations should be used? What is the purpose of adding LF and LB

primers

Answer 1

(A) LAMP is the loop mediated isothermal amplification. It does not need any thermocycler as it amplifies the DNA at constant temperature ranging from 60-65o celcius. Distinct primers are used to amplify different regions.It is very cost effective and easy technique which uses less time and amplify more dna than PCR.

The primers designed for 6 different sites are

In target sequence the 23-25 nt sequence inner to the ends are called F2c and B2. And 40nt inner to the F2c and B2 there are again 23-24 nt sequences named as F1c and B1 and to the outside of ends the sequences designated as F3c and B3. And on the other strand ther are named as B1c,B2c and B3c and F1, F2 and F3 as shown in the followng figure

1. FIP- Forward inner primer - the 3' end of this primer is complementary to the F2 sequence and 5' end is complementary to the F1c of the target sequence.

2. BIP-Backward inner primer - The 3'end is complementary to the B2 region and 5' end to the B1c region of the target sequence

3. FOP- Forward Outer Primer-This is a short lenght primer complementary to the F3 region. It is used in less concentration as compared to the FIP

4. BOP- Backward outer Primer- It is complementary to the B3 region of the template.

5. LF-Forward loop primer- Bind to the forward stem loop

6. LB- Backward loop primer- complementary to the backward stem loop

(B) Designing of primer

The primer can be designed using the primer explorer software considering the following points in mind

1. Specify them in 5' to 3' orientation 2.

2. The GC content should be between 50-60%

3. primers should not form secondary structures

4. avoid repeats of dinucleotide or of the long rns of same nucleotide

5. the primers should not be complementary to each other

6. ends of primer should be stable

(C) The role of primers at each stage of reaction

1. The FIP binds to the target DNA and initiates the synthesis of complementary strand

2. Then FOp hybridizes and extends and replaces the FIP

3. Then BIP binds to the target strand and initiates the synthsis of complementary strand and opens the loop at the 5' end

4.Now BOP hybridizes then extends and replace the BIP

5.At the 3' end then necleotides are added and the DNA forms the stem loop structure and next cycle initiates

(D) range of Tm for each primer

1. FIP-59-63oC

2.BIP-59-63oC

3.FOP-57-61oC

4.BOP- 57-61oC

5.LF- 63-65oC

6.LB- 63-65oC

(E) The relative concentrations used are

The both the outer primers the concentration is 10 times less than the inner primer. For example if FIP and BIP are 50microM then FOP and BOP will be 5 microM

the loop primes are 2 times less than the inner primer.

(F) Purpose of addinf LF and LB is to increase the specificity, efficeincy and accelerate amplification.