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Question about BSP primer design? - What is the complementary strand for forward primer? (Feb/14/2007 )

I am aiming to get the promoter methylation profiling of NPY gene in humans.
But I am doubt about the primer design.

The interested region and primers designed by methyl primer express:

INITIAL NUCLEOTIDE SEQUENCE

AGGACTAGACGGGGCGTGAAGGAAAGAAGGAAAGAAGGAAAGCAGGGATCGGGCACTGCCCGAGGGCAGATACTTGGGCT
TTGGTGTTGTCCAGCGCGCTCGGAGTGCGCTGCCTCGCTCACGCGGTCCCAGGCCCCGCTTCTTCAGGCAGTGCCTGGGGC
GGGAGGGTTGGGGTGTGGGTGGCTCCCTAAGTCGACACTCGTGCGGCTGCGGTTCCAGCCCCCTCCCCCCGCCACTCAGGGGCGGGAAGTGGCGGGTGGGAGTCACCCAAGC
GTGACTGCCCGAGGCCCCTCCTGCCGCGGCGAGGAAGCTCCATAAAAGCCCTGTCGCGACCCGCTCTCTGCACCCCATCCGCTGGCTCTCACCCCTCGGAGACGCTCGCCCGACAGCATAGTACTTGCCGCCCAGCCACGCCCGCGCGCCAGCCACCGTGAGTGCTA
CGACCCGTCTGTCTAGGGGTGGGAGCGAACGGGGCGCCCGCGAACTTGCTAGAGACGCAGCCTCCCGCTCTGTGGAGCCCTGGGGCCCTGGGATGATCGCGCTCCACTCCCCAGCGGACTATGCCGGCTCCGCGCCCCGACGCGGACCAGCCCTCTTGGCGGCTAAATTC
CACTTGTTCCTCTGCTCCCCTCTGATTGTCCACGGCCCTTCTCCCGGGCCCTTCCCGCTGGGCGGTTCTTCTGAGTTACCT
TTTAGCAGATATGGAGGGAGAACCCGGGA

BISULFITE MODIFICATION OF DNA

CGCGTTCGGAGTGCGTTGTTTCGTTTACGCGGTTTTAGGTTTCGTTTTTTTAGGTAGTGTTTGGGGCGGGAGGGTTGGGGTGTGGGTGGTTTTTTAAGTCGATATTCGTGCGGTTGCGGTTTTAGTTTTTTTTTTTCGTTATTTAGGGGCGGGAAGTGGCGGGTGGGAGTTATTTAAGC
GTGATTGTTCGAGGTTTTTTTTGTCGCGGCGAGGAAGTTTTATAAAAGTTTTGTCGCGATTCGTTTTTTGTATTTTATTCGTTGGTTTTTATTTTTCGGAGACGTTCGTTCGATAGTATAGTATTTGTCGTTTAGTTACGTTCGCGCGTTAGTTATCGTGAGTGTTAC
GATTCGTTTGTTTAGGGGTGGGAGCGAACGGGGCGTTCGCGAATTTGTTAGAGACGTAGTTTTTCGTTTTGTGGAGTTTTGGGGTTTTGGGATGATCGCGTTTTATTTTTTAGCGGATTATGTCGGTTTCGCGTTTCGACGCGGATTAGTTTTTTTGGCGGTTAAATTT
TATT

FORWARD

Length: 21bp.
5' GGTGTGGGTGGTTTTTTAAGT 3'
Tm=61.36; CpG=0; C=4
You may modify the primer sequence if necessary, within this region:
5' GGGYGGGAGGGTTGGGGTGTGGGTGGTTTTTTAAGTYGATATTYGTGYGGT 3'


REVERSE

Length: 18 bp.
5' CAAAACCCCAAAACTCCA 3'
Tm=61.15; CpG=0; C=6
You may modify the primer sequence if necessary, within this region:
5' AAAACRCRATCATCCCAAAACCCCAAAACTCCACAAAACRAAAAACTA 3'


PCR PRODUCT

Length: 358 bp.
5' GGTGTGGGTGGTTTTTTAAGTYGATATTYGTGYGGTTGYGGTTTTAGTTTTTTTTTTTYGTTATTTAGGGGYGGGAAGTG
GYGGGTGGGAGTTATTTAAGYGTGATTGTTYGAGGTTTTTTTTGTYGYGGYGAGGAAGTTTTATAAAAGTTTTGTYGYGAT
TYGTTTTTTGTATTTTATTYGTTGGTTTTTATTTTTYGGAGAYGTTYGTTYGATAGTATAGTATTTGTYGTTTAGTTAYGT
TYGYGYGTTAGTTATYGTGAGTGTTAYGATTYGTTTGTTTAGGGGTGGGAGYGAAYGGGGYGTTYGYGAATTTGTTAGAGA
YGTAGTTTTTYGTTTTGTGGAGTTTTGGGGTTTTG 3'

%CGs=41.62


My question is:
Firstly,we look the orignial sequence for forward primer and it's complementary strand sequence:
5'-GGTGTGGGTGGCTCCCTAAGT-3'
3'-CCACACCCACCGAGGGATTCA-5'
After bisulfite treated,they become:
5'-GGTGTGGGTGGUTUUUTAAGT-3'
3'-UUAUAUUUAUUGAGGGATTUA-5'
They are not complementary any more.
Then, if we use the forward primer: 5' GGTGTGGGTGGTTTTTTAAGT 3' to amplify,but it will not complementary to 3'-UUAUAUUUAUUGAGGGATTUA-5',then what kind of sequence the forward primer will bind to(or complementary to it)?I cannot find this kind of sequence,so I cannot figure out how the forward primer will be extension.Could anyone explain it?

Thanks a lot!

-sallynie-

In the first round of PCR, only the reverse primer has a target - the modified forward strand. In the second round, the newly synthetized complementary strand to the forward strand contains the target of the forward primer. In BSP, you need primer pairs for each strand, as they, as you rightly mentioned, are no longer complimentary. INmost cases, people look only at one strand and often this is the forward strand, however, there are also papers reporting both strands (that meens, you double the work wink.gif) or only the reverse strand.

To make it clear (very short sequences - would never work in real life):
Your primers have the sequence: F: 5'-TTTTAGT-3' and R: 5'-AATCAAAT-3'

Unmodified:
5'- CTCTAGTGCGTCGTAGAAATTGACT-3'
3'- GAGATCACGCAGCATCTTTAACTGA-5'

After bisulfite:
5'- TTTTAGTGYGTYGTAGAAATTGATT-3'

3'- GAGATTATGYAGYATTTTTAATTGA-5'

First PCR cycle: Annealing

5'- TTTTAGTGYGTYGTAGAAATTGATT-3'
......................................TAAACTAA Primer R binds (the dotted line was only needed to move the primer to the right postition)

Extension
5'- TTTTAGTGYGTYGTAGAAATTGATT-3'
3'- AAAATCACRCARCTCTTTTAACTAA-5'

Second PCR cycle: Denaturation + Annealing

5'- TTTTAGTGYGTYGTAGAAATTGATT-3'
......................................TAAACTAA
......TTTTAGT....................................Primer F binds
3'- AAAATCACRCARCTCTTTTAACTAA-5'



Everything clear now?

Good luck,

Krümel

-krümelmonster-

Thank you very much, Krümel !
Is this kind of PCR strand specific amplification?
Why it avoid to design the former primer complementary to the converted complementary strand just as the way we design the reverse primer?
What is the advantage of strand specific amplification compared to regular PCR?

Thanks!

-sallynie-

QUOTE (sallynie @ Feb 15 2007, 04:29 PM)
Thank you very much, Krümel !
Is this kind of PCR strand specific amplification?

Yes, it has to be, as you don't have complimentary strands after bisulfite treatment.
QUOTE (sallynie @ Feb 15 2007, 04:29 PM)
Why it avoid to design the former primer complementary to the converted complementary strand just as the way we design the reverse primer?
What is the advantage of strand specific amplification compared to regular PCR?


Well, if you design primers like you do in "normal" PCR, you will have one primer binding to the forward and one binding to the reverse strand. But then, you don't have primers binding to the newly synthetized strands and the exponential amplification won't take place.
Okay, you may now ask why we don't find primer sequences that are completely unchanged by Bisulfite and should therefore be present in the forward strand and in the complementary strand synthetized along the reverse strand. - It would be difficult to find sequences that can be used as primer binding sites that are not modified by bisulfite. But even if you would find such primers - you would still end up with two different PCR products making sequencing impossible.
So, to make a long story short - the advantage of strand specific amplification for bisulfite modified DNA is that this is the only way amplification can work!

K.

-krümelmonster-