Variant (E484K) ALERT - Ligation-Dependent Loop-Mediated Isothermal Amplification

Oligonucleotides used for RNA templated DNA ligation using

All sequences shown 5' to 3. Underlined segments are complementary to the target and bold nucleotide is specific to the G>A substitution at position 23012 resulting in the E484K amino acid change.

E484K-Donor

/5Phos/ AACACCATTACAAG CTACGTACATGACATAATCCAAACTCATAAATTCTCGCATTTTAGATCCGTCTCCTTACAGGACACATCATCC

E484-Acceptor

CATCTCTAACTCTACTAAGACTTCCATTTATCAACAATAGCTGACATGTTCTAGCAGCGACAGGACACACGACAGG AGGAAAGTAACAATTAAAACCTTT T

Prepare ligation mix to use with previously isolated RNA. A standard Trizol/Chloroform method or column purification method for RNA isolation works well.

X1 Ligation mix (multiply for number of samples processed)

2µL E484K-Donor (100nM)

2µL E484K-Acceptor (100nM)

1µLSplintR Buffer (NEB)

0.2µL (NEB)

1.8µL molecular biology grade water

• add 3µL RNA per 7µL of reaction mix for a 10µL reaction volume

Incubate at 37°C for 0h 20m 0s followed by a 0h 20m 0s at 65°C

inactivation step.

Place ligation reactions on ice.

Oligonucleotides used for Loop-Mediated Isothermal Amplification

all sequences shown 5' to 3'

FIP

CTTTCCTCCTGTCGTGTGTCCTCCATTTATCAACAATAGCTGAC

BIP

ACCTTTAACACCATTACAAGCTACGACGGATCTAAAATGCGAGAA

F3

CATCTCTAACTCTACTAAGACT

B3

GGATGATGTGTCCTGTAAGG

Prepare LAMP mix.

X1 LAMP mix (multiply for number of samples processed)

2.5µL Isothermal Buffer 1 (NEB)

0.5µL (100mM)

3.5µL dNTPs (10µM)

0.8µL FIP (50µM)

0.8µL BIP (50µM)

0.5µL F3 (10µM)

0.5µL B3 (10µM)

0.1µL (100µM)

1µL

11.8µLMolecular Biology Grade water

• add 3µL of the ligation product from the Ligation step to 22µL of LAMP mix for a 25µL reaction.

Incubate at 65°C for 0h 50m 0s

Visualize reaction tubes over a UV transilluminator. Samples containing a G>A mutation at position 23012 will exhibit fluorescence.