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A small confusion about Illumina Amplicon Sequencing Protocol
Hello everyone!
I am new to this forum. I will be working very shortly on Illumina MiSeq. So, I am currently trying to understand a protocol for Amplicon Sequencing but facing some confusion in one area. So any help will be appreciated.
The protocol describes a 2 step PCR process:
The first round PCR involves designing and optimizing a primer set that (i) is specific to your target sequence, and (ii) contains a universal or common adapter sequence for the 2nd round index primers to bind.
So the confusion is that how come a universal or common adapter sequence from second round PCR primers will bind to first round PCR primer's common adapter sequences since they are not complimentary but common sequences.
PRIMERS FOR 1st ROUND PCR
Primer1: Illumina adapter + forward target primer
5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -forward-primer-3’
Primer2: Illumina adapter + reverse target primer
5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -reverse-primer-3’
In the second round PCR, the adapters are extended to include the index sequences and flow-cell attachment regions
PRIMERS FOR 2nd ROUND PCR
AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC
CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG
The bold letters indicate Index sequences and underlined show the sequence which is common with with small region of adapter of first round target specific primers
So the confusion arises about how binding of this underlined adapter sequence with first round's PCR primers(having small sequence of adapter regions) is possible since they are no complimentary to each other but are common sequences.
Here is the link for that protocol for any reference!!
Kindly guide me about it. Thanks a lot
I am new to this forum. I will be working very shortly on Illumina MiSeq. So, I am currently trying to understand a protocol for Amplicon Sequencing but facing some confusion in one area. So any help will be appreciated.
The protocol describes a 2 step PCR process:
The first round PCR involves designing and optimizing a primer set that (i) is specific to your target sequence, and (ii) contains a universal or common adapter sequence for the 2nd round index primers to bind.
So the confusion is that how come a universal or common adapter sequence from second round PCR primers will bind to first round PCR primer's common adapter sequences since they are not complimentary but common sequences.
PRIMERS FOR 1st ROUND PCR
Primer1: Illumina adapter + forward target primer
5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -forward-primer-3’
Primer2: Illumina adapter + reverse target primer
5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -reverse-primer-3’
In the second round PCR, the adapters are extended to include the index sequences and flow-cell attachment regions
PRIMERS FOR 2nd ROUND PCR
AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC
CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG
The bold letters indicate Index sequences and underlined show the sequence which is common with with small region of adapter of first round target specific primers
So the confusion arises about how binding of this underlined adapter sequence with first round's PCR primers(having small sequence of adapter regions) is possible since they are no complimentary to each other but are common sequences.
Here is the link for that protocol for any reference!!
Kindly guide me about it. Thanks a lot
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