Fusion primer and nested integrated PCR (FPNI-PCR)

Protocol for Tail-PCR/ FPNI-PCR: 

Round1: 

10x LA buffer – 1ul

MgCl2 – 1ul

dNTP (2.5mM) – 1.6ul

F primers – 4ul

              (Separate PCR for each of the FP primers 1-9)

R primer (gene-specific) – 0.8ul

DNA (100ng/ul) – 1.5ul

LA Taq – 0.1ul

H₂O – 0ul

Total Volume=10ul

Round1 with DMSO: (This method gave better results. Gpa1 has a high GC content)

10x LA buffer – 1ul

MgCl₂ – 1ul

dNTP (2.5mM) – 1.6ul

F primers – 4ul

              (separate PCR for each of the FAD primers 1-9.)

R primer (gene-specific) – 0.8ul

DNA (100ng/ul) – 1.3ul

DMSO – 0.2ul

LA Taq – 0.1ul

H₂O – 0ul

Total Volume=10ul

Round1 Cycle: 

1. 95 ^OC - 1:30
2. 94 ^OC - 0:10
3. 62 ^OC – 0:30
4. 72 ^OC - 2:00
5. 94 ^OC – 0:15
6. 25 ^OC – 1:00
7. 28 ^OC, increase 0.2^OC/sec – 3:00
8. 72 ^OC – 2:30
9. Go To Step 2 5x (total of 6x)
10. 94 ^OC – 0:10
11. 62 ^OC – 0:30
12. 72 ^OC C – 2:00
13. Go To Step 10 1x (total 2x)
14. 94 ^OC – 0:10
15. 44 ^OC – 0:30
16. 72 ^OC – 2:00
17. 72 ^OC – 5:00
18. 10 ^OC - forever

Round 2: 

Dilute Round1 PCR product ½ to use as template. Still keep the FP samples 1-9 separate.

(Note: Most genes will need 5x HF buffer and no DMSO. Gpa1 contains high-GC regions though)

5x GC Rich Phusion Buffer – 4ul

dNTP (2.5mM) – 1.6ul

FSP1 – 4ul

Gene specific R (nested) – 1.6ul

DMSO – 0.6

½ conc. PCR template – 1ul

Phusion – 0.2ul

H₂O – 4.6ul

Cycle conditions: 

1. 98 ^OC – 1:30
2. 98 ^OC – 0:10
3. 61 ^OC – 0:30
4. 72 ^OC – 1:30
5. Go To step 2 34x (total of 35x)
6. 72 ^OC – 5:00
7. 10 ^OC – forever

Round 3: 

Dilute Round2 PCR product 1/40 to use as template. Still keep the FP samples 1-9 separate.

(Note: Most genes will need 5x HF buffer and no DMSO. Gpa1 was a high-GC content region though)

5x GC Rich Phusion Buffer – 4ul

dNTP (2.5mM) – 1.6ul

FSP2 – 4ul

Gene specific R (nested) – 1.6ul

DMSO – 0.6

1/40 conc. PCR template – 1ul

Phusion – 0.2ul

H2O – 4.6ul

Cycle conditions: 

1. 98 ^OC – 1:30
2. 98 ^OC – 0:10
3. 64 ^OC – 0:30
4. 72 ^OC – 1:30
5. Go To step 2 34x (total of 35x)
6. 72 ^OC – 5:00
7. 10 ^OC – forever

Final steps:

Run all 9 samples on 1% agarose gel. Use a gel extraction kit to excise and purify the major bands. Send for sequencing.

Primers: 

Will need 3 gene specific primers, each nesting closer to the unknown sequence region.

9 different FP primers, and 2 “nesting” FSP primers: 

Acronyms: 

FP = “Fusion arbitrary degenerate primers”, also called FAD primers.

FSP = “FP-specific primers”

Please refer to Wang et al. (2011).
Reference:
Wang Z, Ye S, Li J, Zheng B, Bao M, Ning G. Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotechnol. 2011;11:109.