TAIL-PCR Protocol

TAIL-PCR protocol

Honggui La

PART I TAIL-PCR protocol

TAIL is a series of reactions that are intended to map where a T-DNA (transfer DNA) has inserted within the genome. The main components of the 3 reactions are the AD (Arbitrary Degenerate) primers, border primers, and DNA from the T-DNA lines that are to be mapped. AD primers are degenerate primers that anneal throughout the genome. The border primers are specific for the left and right borders of the T-DNA. From the primary reaction to the tertiary, the border primers get closer to the edge of the T-DNA. That is why a 'shift' is visible when running a gel with the secondary and tertiary reactions next to each other. The success rate of TAIL-PCR varies, depending onhow many DNA samples, AD primers, and border primers are used.

1. Dilute the DNA sample 1:5 (Dilute more or less depending on DNA concentration.)
2. Add 5µL DNA, and 5µL AD primers to PCR plate according to the diagram below (each AD primer has a specific concentration, see Additional Information at the end of the protocol):
   NOTE: Keep plate on ice throughout the procedure.

Note:
DNA1, DNA2, DNA3, ... = Individual DNA samples for T-DNA mapping. Add 5µL DNA (1° reaction) in an entire horizontal row (e.g. A) for each individual.
AD1, AD2, AD3, ... = Arbitrary Degenerate primers. Add 5µL of the 4X AD primer (1° reaction) to each vertical column as diagram indicates.
lightyellow= Left half of plate-Add LB1 primer cocktail.
grey=Right half of plates-Add RB1 primer cocktail.

1. Start the 1° Reaction (detailed in Additional Information) program on thermal cycler and press PAUSE, letting the block cool to 4°C.
2. Mix the LB1 and RB1 cocktails according to TAIL Recipeincluded.
   NOTE: Add Taq polymerase last.
3. Add 10µL of each cocktail (LB1 and RB1) to appropriate wells according to previous diagram.
4. Place plate in thermal cycler and press PAUSE, again to allow the reaction to proceed.
5. To prepare the 2° reaction, dilute 1° TAIL reaction 200-fold by transferring 1µL PCR products to 199µL ddH2O. (This is most easily achieved through the use of a multi-channel pipette.)
6. Set up 2° reaction plate according to same diagram, except use 4µL diluted DNA. NOTE: As before, keep plate on icethroughout preparation.
7. Add 5µL of the AD primers to the appropriate wells.
8. Start 2°reaction program on thermal cycler and press PAUSE.
9. Add 11µL of border (LB2 or RB2) cocktail to appropriate wells and place plate in thermal cycler. Press PAUSE to allow reaction to proceed.
10. Once the 2° reaction has completed, the products can either be sequenced or a 3° reaction can be run to further purify the PCR products if there are many nonspecific products. CONTINUE if a 3° reaction is needed. To prepare samples for sequencing, SKIP to step 25.
11. The 3° reaction is prepared like the 2°. 2° reaction productneeds to be diluted 100-fold and the overall reaction volume is 50µL. Add the diluted products from the 2° reaction to a new PCR plate. Again, keep reaction on ice and use a multi-channel pipette for diluting.
12. Add 12.5µL of the AD primers to the appropriate wells.
13. Start the 3° reaction program on the thermal cycler and press PAUSE.
14. Mix the LB3 and RB3 cocktail (adding the Taq last) and add 32.5µL to appropriate half of plate.
15. Flash spin in a tabletop centrifuge to assure all reaction contents are at the bottom of the wells.
16. Place plate in thermal cycler and press PAUSE again to allow reaction to proceed.
17. To sequence entire contents of plate, SKIP to step 25. To run a gel and visualize the 3°reactions, go to these steps: Prepare a large 1% agarose gel with 4 rows of 50 wells (200 total wells).
18. Add the appropriate ladder (100bp or 1kb) to the first and last well in each row.
19. Using a multi-channel pipette, draw 7µL from row A or the 2° reaction. Expel this amount on a piece of parafilm. Using the same pipette tips, draw 3µL of loading dye and add it to the droplets on the parafilm. Mix the dye and reaction contents by pipetting up and down.
20. Without changing tips, draw all 10µL of the samples and add them to the gel starting next to the ladder in the top, left portion of the gel. NOTE: Using the multi-channel pipette will leave a space between the samples, this isdesired.
21. Discard the pipette tips and repeat previous step until entire 2° reaction contents are loaded into the gel. Assure a space is left between all 2° reactions added to gel.
22. Now, do the same with the 3° reactions, add the 10µL of the 3° reactions directly next to the 2° reactions. If loaded properly, all lanes will be filled without spaces. This will make the gel easier to analyze. There should be a visible shift in product length from the 2° to the 3° reaction. If there are multiple bands visible in one lane, purify individual bands for sequencing via the Topo Cloning Procedure. If single bands exist in the 3° reaction, continue to step 25 for product purification.
23. The PCR products must be purified before they can be sequenced. This can be done individually via the Qiagen PCR purification protocol. After that, the purified DNA samples can be sequenced directly or be cloned into T-vector for sequencing.

TAIL 1° REACTION PROGRAM:

Control Method: CALCULATED

1=4° for 2 min.

2=93° for 1 min.

3=95° for 1 min.

4=94° for 30 sec.

5=62° for 1 min.

6=72° for 2 min. 30 sec.

7=Go to step 4 for 4 more cycles

8=94° for 30 sec.

9=25° for 3 min.

10=Ramp for 72° at 0.2°/sec, 72° for 2 min. 30 sec.

11=94° for 10 sec.

12=68° for 1 min.

13=72° for 2 min. 30 sec.

14=94° for 10 sec.

15=68° for 1 min.

16=72° for 2 min. 30 sec.

17=94° for 10 sec.

18=44° for 1 min.

19=72° for 2 min. 30 sec.

20=Go to step 12, for 14 more cycles

21=72° for 5 min.

22=4° forever

23=END
TAIL 2° REACTION PROGRAM:

Control Method: CALCULATED

1=4° for 2 min.

2=94° for 10 sec.

3=64° for 1 min.

4=72° for 2 min. 30 sec.

5=94° for 10 sec.

6=64° for 1 min.

7=72° for 2 min. 30 sec.

8=94° for 10 sec.

9=44° for 1 min.

10=72° for 2 min. 30 sec.

11=Go to step 2, for 11 more cycles

12=72° for 5 min.

13=4° forever

14=END
TAIL 3° REACTION PROGRAM:

Control Method: CALCULATED

1=4° for 2 min.

2=94° for 10 sec.

3=44° for 1 min.

4=72° for 2 min. 30 sec.

5=Go to step 2, for 19 more cycles

6=72° for 5 min.

7=4° forever

8=END
AD (Arbitrary Degenerate) Primer Sequences and Concentrations:

Stock concentrations of AD primers should be 20µM. To achieve the concentrations required for TAIL reactions, dilute in a seperate tube. The final amount of 400µL is sufficient for all 3 TAIL reactions.
64-fold degenerate Add 160µL primer and 240µL ddH2O
128-fold degenerate Add 240µL primer and 160µL ddH2O
256-fold degenerate Add 320µL primer and 180µL ddH2O

Pipette this: 10 µl/rxn

Total rxn = 20 µL

5 µL of diluted template (Add 400 µLH2O to 100µL DNA)

5 µL 4X AD primer

10 µL master mix

Pipette this: 11 µL/rxn

Total rxn =20 µL

4 µL of 200 fold diluted primary product

5 µL 4X AD primer

11 µL master mix

Pipette this: 32.5 µL/rxn

Total rxn =50 µL

5 µL of 100 fold diluted secondary product

12.5 µL 4X AD primer

32.5 µL master mix

PART II How to design primers to confirm Tos17 insertion lines

1. Judge the insertion orientation of Tos17

Because only 3’ terminal flanking sequences are sequenced, we can just use one sequence for BLAST search against databases. If you find the sequence matches plus strand of certain gene, it means Tos17 inserts into this gene forwardly; otherwise, Tos17 inserts reversely (see the following figures and BLAST results).

Forward insertion

LOC_Os03g12570|11973.t01056|unspliced-genomic C-5 cytosine-specific DNA

methylase family protein, expressed

Length = 10,798

Plus Strand HSPs:

Score = 1420 (219.1 bits), Expect = 5.5e-58, P = 5.5e-58

Identities = 284/284 (100%), Positives = 284/284 (100%), Strand = Plus / Plus

Query: 1 CAAGAGACGGTTGTTAAATCTTCGGCAGGAACTCCTGGAAGTGAAATAGCTGATTCGGAA 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 5700 CAAGAGACGGTTGTTAAATCTTCGGCAGGAACTCCTGGAAGTGAAATAGCTGATTCGGAA 5759

Query: 61 CAGTTGTTTGAATAGTATGTTATCTGAGTCACTTGTATAGTTGGGTGATGTGCTATAATT 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 5760 CAGTTGTTTGAATAGTATGTTATCTGAGTCACTTGTATAGTTGGGTGATGTGCTATAATT 5819

Query: 121 GTTTCTGTATGAGTTTCGAACACGGACAGTGCATGCATTGTCCAATGCTCCAAACATTCA 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 5820 GTTTCTGTATGAGTTTCGAACACGGACAGTGCATGCATTGTCCAATGCTCCAAACATTCA 5879

Query: 181 CTGGTTAACCCTGGAACTGAGTAATTTGCATACGTTTCTTCATTGGATGTCCATTTCTTT 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 5880 CTGGTTAACCCTGGAACTGAGTAATTTGCATACGTTTCTTCATTGGATGTCCATTTCTTT 5939

Query: 241 CTGGTTTAGATATGTGAGTGAAATACTTTATTTGCTTCGATTCT 284

||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 5940 CTGGTTTAGATATGTGAGTGAAATACTTTATTTGCTTCGATTCT 5983

Reverse insertion

LOC_Os03g58400|11973.t05655|unspliced-genomic expressed protein

Length = 2245

Minus Strand HSPs:

Score = 1720 (264.1 bits), Expect = 6.0e-73, P = 6.0e-73

Identities = 344/344 (100%), Positives = 344/344 (100%), Strand = Minus / Plus

Query: 344 GGGCAATTATGGACAAGTGTGGTGATTCTGATGATTGCATCTCCACTTCTGAGGCTGCTG 285

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 194 GGGCAATTATGGACAAGTGTGGTGATTCTGATGATTGCATCTCCACTTCTGAGGCTGCTG 253

Query: 284 AACGAGCAGCTAAACTTTCTGAGGACAAGATTAAGAATCTGCCCGTGCCTGGCGAAGTAG 225

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 254 AACGAGCAGCTAAACTTTCTGAGGACAAGATTAAGAATCTGCCCGTGCCTGGCGAAGTAG 313

Query: 224 AATTCATAAATGGTGGCCCTCCGTGTCAGGTCAGTTGCTATGTGGCTTTTGCCTGTATAC 165

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 314 AATTCATAAATGGTGGCCCTCCGTGTCAGGTCAGTTGCTATGTGGCTTTTGCCTGTATAC 373

Query: 164 CAGGGAGCTCCTAACAACACATTCGACATTGCAAGCCAATTGCTTGACCTTTTGACCTAT 105

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 374 CAGGGAGCTCCTAACAACACATTCGACATTGCAAGCCAATTGCTTGACCTTTTGACCTAT 433

Query: 104 CCTTTTTTAGGGTTTTTCTGGGATGAACAGATTCAATCAAAGTCCCTGGAGCAAAGTCCA 45

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 434 CCTTTTTTAGGGTTTTTCTGGGATGAACAGATTCAATCAAAGTCCCTGGAGCAAAGTCCA 493

Query: 44 GTGCGAGATGATCTTAGCATTCCTGTCATTTGCGGAGTATTTCC 1

||||||||||||||||||||||||||||||||||||||||||||

Sbjct: 494 GTGCGAGATGATCTTAGCATTCCTGTCATTTGCGGAGTATTTCC 537

1. Design of primers

Once the insertion orientation is known, the whole gene sequences will be downloaded and used for primer design. If forward insertion, 3’ terminal downstream sequences of the gene will be used to design 3’ terminal primer. Then the primer will be used along with 3’ terminal primer anchored on Tos17 to isolate 3’ terminal flanking sequence. Minus strand of this gene will be used to design 5’ terminal primer. If reverse insertion, 3’ terminal downstream sequences of minus strand of the gene will be used to design 3’ terminal primer. Plus strand of this gene will be used to design 5’ terminal primer.

1. PCR amplification

Normal PCR will be performed to amplify flanking sequences. There are three primer combinations for use, 3’ universal primer/3’ gene-specific primer, 5’ universal primer/5’ gene-specific primer, 3’ gene-specific primer/5’ gene-specific primer. The PCR bands will be recovered and sequenced to confirm.