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Old 01-09-2011, 10:53 PM   #1
seqgirl123
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Default how does index-tag PCR work?

In normal PCR, you have your two PCR primers that flank the target region, and the target region is amplified from the genomic DNA after 30 cycles of PCR. That is standard for most cases.

I am trying to understand how index PCR works where one of the two PCR primers contains a index-tag (say a 6bp tag), and you go ahead and PCR amplify your target region from genomic DNA with 30 cycle PCR like normal, but the index tag remains in the final PCR product along with your target region. How does the index still remain in the final PCR product, while in the first case above for normal PCR, your primer sequences just anneal but don't remain as part of your final target region? How is it that in this index-PCR, the primer sequence with the index tag remains..how is this PCR done?

I post it in this forum because I know Illumina sequencing does a lot of this where it reads through an index sequence. I understand how the index is read and sequenced, but what I don't understand how the PCR purified product contains an index tag in the final PCR. Does anyone know?
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Old 01-10-2011, 06:22 AM   #2
kmcarr
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Default

seqgirl,

See the attached PDF for a pictorial representation of the described steps.

1. Fusion primer A which contains target specific (blue), MID tag (yellow) and sequencing platform specific (green) portions is partially annealed to its target in genomic DNA. It is extended past the target site for the second PCR primer.

2. Fusion primer B which contains target specific (red) and sequencing platform specific (green) portions is partially annealed to the product of step 1.

3. Extension from primer B terminates at the end of its template strand.

4. Primer A fully anneals to the 5' end of of the newly synthesized strand from step 3.

5. Extension from primer A terminates at the end of its template strand.

From this point forward primers A and B fully anneal to the product of previous PCR rounds, producing fragments which look like the product in step 5.

The orange arrow indicates the position where you sequencing primer would anneal, showing how the MID tag would constitute the 5' end of your read.
Attached Files
File Type: pdf AmpliconFusionPrimers.pdf (71.3 KB, 233 views)
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Old 01-10-2011, 10:33 AM   #3
Bruce E
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Default Index PCR

Your original thought is incorrect. In all PCR the pcr primers become part of your product. The only difference with index pcr is that you add an index sequnce to the 5' end of your pcr primer.
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