Apr 15, 2025

Public workspaceMeasles virus whole genome amplification (D4, B3,D8 genotypes) by in-house tiling-PCR V.2

DOI
dx.doi.org/10.17504/protocols.io.rm7vz63wxgx1/v2
  • Nora Deezsi-Magyar1,
  • Zita Rigó1
  • 1National Center for Public Health and Pharmacy
Nora Deezsi-Magyar
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DOI: dx.doi.org/10.17504/protocols.io.rm7vz63wxgx1/v2
Protocol Citation: Nora Deezsi-Magyar, Zita Rigó 2025. Measles virus whole genome amplification (D4, B3,D8 genotypes) by in-house tiling-PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz63wxgx1/v2Version created by Nora Deezsi-Magyar
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 15, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 126708
Abstract
The protocols allows whole genome amplification of the Measles virus Genotypes D4, B3 and D8 by utilizing tiling-PCR and in-house designed primer sets in two pools.
DNAse I treatment (to remove contaminating human-derived gDNA)
DNAse I treatment (to remove contaminating human-derived gDNA)
1h 20m
1h 20m
Reagents
  • TURBO DNase‱ (2 U/μL), Invitrogen
  • Nuclease-free destillated water
Prepare the following master mix
AB
TURBO DNase (2 U)1.0 uL
TURBO DNase puffer4.0 uL
Destillated water6.0 uL
RNA40.0 uL

Incubation in thermocycler
Temperature37 °C Duration00:30:00

Post-DNAse I treatment clean-up

Reagents
  • AMPure XP Beads, Beckman Coulter
  • 70% EtOH (prepared freshly)
  • Nuclease-free destillated water
Add 1.8 μl AMPure XP per 1.0 μl of sample.
Bind DNA fragments to paramagnetic beads.
Separation of beads + DNA fragments from contaminants.
Wash beads + DNA fragments twice with 70% EtOH to remove contaminants.
Elute purified DNA fragments from beads.
Transfer to new tubes.
Reverse transcription
Reverse transcription
1h 30m
1h 30m
Reagents
  • SuperScript‱ VILO‱ cDNA Synthesis Kit, Invitrogen
  • Nuclease-free destillated water

Prepare the following master mix
AB
5X VILO™ Reaction Mix4.0 uL
10X SuperScript™ Enzyme Mix2.0 uL
Destillated water4.0 uL
RNA8.0 uL


Incubation in thermocycler
Temperature25 °C Duration00:10:00

10m
Incubation in thermocycler
Temperature42 °C Duration01:00:00

1h
Incubation in thermocycler
Temperature85 °C Duration00:05:00

5m
Polymerase chain reaction (tiling-PCR)
Polymerase chain reaction (tiling-PCR)
6m 50s
6m 50s
Reagents
  • Phusion‱ High-Fidelity DNA Polymerase, New England Biolabs
  • Nuclease-free destillated water
  • dNTP mix
Prepare the following master mixes

Mix_1
AB
5x Phusion™ High-Fidelity PCR Master Mix with GC Buffer4.0 uL
dNTP mix (10 mM)0.4 uL
Phusion™ High-Fidelity DNA Polymerase0.2 uL
Primer pool_13.0 uL
Destillated water7.4 uL
cDNA template5.0 uL
The mix includes pool_1 primers (see section 4.2)

Mix_2
AB
5x Phusion‱ High-Fidelity PCR Master Mix with GC Buffer4.0 uL
dNTP mix (10 mM)0.4 uL
Phusion‱ High-Fidelity DNA Polymerase0.2 uL
Primer pool_23.0 uL
Destillated water7.4 uL
cDNA template5.0 uL
The mix includes pool_2 primers (see section 4.2)
Tiling-PCR primers
StartEndPool nrForward/ReverseSequence (5'-3')
1291FCCAAACAAAGTTGGGTAAGGATAGATCA
110111251RCTACTCCCATGGCATAGCTCCATA
106610912FTCAGAACAAGTTCAGTGCAGGATCA
216621902RAGAGTCAGCATCTTGGATTCCCTT
209421181FTCAAGAAATCTCCAGGCATCAAGC
319432211RCGACTGGATTTTATAATGGAGCGGATT
313531572FAAAAAGATGAGCTCAGCCGTCG
423542582RAGTTGTGCATGGAGAGTCTTGCT
420042241FGCACCAGTCTTCACATTAGAAGCA
530053221RGCTGTGGGATGCTGATGTCGTT
525752772FAACCAGCACCCAAGAGCGAT
635763792RAGCCTATGTTGTACGAGACCCC
631863411FCTGTCCGAGATTAAGGGGGTGAT
741874381RTGCAATGGCTAGCAACCCGA
738374072FGCTGGCTGTTCTATTCGTCATGTT
848385062RGACCCCGTATGAAGGAATCCTGT
842884521FAAGGGTAAAATCCAAGCACTCTGC
952895541RACGGATCTTCCTTGTTGACTCTTTGT
946594912FGTTATCCGACCCACTCTCATATTCCA
10565105902RGCATAAAGCAGCCAAATCTCACTCC
10533105541FAGCAGTGCGTTGACAACTGGA
11633116641RGCCTTTGCCTAAGAATTACAAAATAATCTCT
11600116232FAACCTTAAGAAACGGGAAGCTGC
12700127262RATTCTAGTACATCAGGGACCTCAAGG
12658126801FCCATATGTGGGCAAGGCTAGCT
13758137811RTGAAGACAACAGCTCACCCATCT
13717137482FGGCATTTGATGTACATTATCATAGACCATCA
14817148472RTGGGGATATTACTGACTATGAAATTGAAGC
14766147881FAGGTGCTCTTTAATGGGAGGCC
15865158911RAGACAAAGCTGGGAATAGAAACTTCG
Tiling-PCR thermocycling
1 Temperature98 °C Duration00:00:30
2 Temperature98 °C Duration00:00:10
3 Temperature52 °C Duration00:00:20
4 Temperature72 °C Duration00:00:50
5 Repeat steps 2-3-4 for 40 times.
6 Temperature72 °C Duration00:05:00
7 Temperature10 °C hold

6m 50s
Post-PCR clean-up

Reagents
  • AMPure XP Beads, Beckman Coulter
  • 70% EtOH (prepared freshly)
  • Nuclease-free destillated water
Add 0.8 μl AMPure XP per 1.0 μl of sample.
Bind DNA fragments to paramagnetic beads.
Separation of beads + DNA fragments from contaminants.
Wash beads + DNA fragments twice with 70% EtOH to remove contaminants.
Elute purified DNA fragments from beads.
Transfer to new tubes.
Tiling-amplicon quantification using Qubit Flex fluorometer
Tiling-amplicon quantification using Qubit Flex fluorometer
10m
10m
Reagents
  • Qubit 1X dsDNA HS Assay Kit
Add 190 uL of working solution to all wells of two Qubit strips. Label the top of the tubes S1 and S2. Then add 10 uL of the respective HS Standard (1 or 2) to each tube of the two strips.
For each of your sample tubes, add 198 uL of working solution to labeled tubes.
Then add 2 uL of your samples to each tube.
Vortex all strips and spin down if necessary. Incubate for 3-5 minutes at room temperature.
On the Qubit, select 1x dsDNA -> dsDNA High Sensitivity -> Read standards
Insert strip standard 1 and then select Read standard. Repeat for standard 2.
Select Run samples and read the respective wells of the strips. Use the 2 uL sample volume. When you are done.