Protocol for foamy vector genome titer



FOAMY VECTOR GENOME TITER (RUSSELL LAB PROTOCOL).

Note that foamy virus (FV) vectors have a DNA genome since they complete reverse transcription in the packaging cell. Therefore one can directly isolate vector DNA from the stock and titer with qPCR as below. This protocol works on all DeltaPhi (ΔΦ) FV vectors (see Trobridge et al., 2002, Mol Therapy, v6 p321).

Materials:

Primers:

Forward primer: 5’-CGCTACTCCCTCTGACATCC

Reverse primer: 5’-TTATATTCGAGCCCCACGTT

Probe: 5’-TACTCGCTGCGTCGAGAGTGTACGAGAC

This is a TaqMan® TAMRA probe synthesized by Applied Biosystems, using 6FAM (6-carboxy fluorescein) as reporter dye and TAMRA is the quencher.

Standard plasmid:

pACT5FVMF(f). This plasmid contains an intact vector provirus of ΔΦMF (internal MLV LTR promoter driving GFP, see Hendrie et al., 2008, Mol Therapy v16 p534). The plasmid sequence follows at the end of this document.

TaqMan( Gene Expression Master Mix from Applied Biosystems (2X)

DpnI (from New England Biolabs)

Pellet Paint DNA carrier non-fluorescent (Novagen)

PCR Machine: StepOnePlus™ Real time PCR machine (Applied Biosystems)

Prepare dilutions of the plasmid pACT5FVMF(f) standard

Make the dilutions below from a clean, phenol/chloroform-extracted and ethanol-precipitated maxiprep of plasmid pACT5MF(f). Dilute with TE.

|Copy # per 5 ul |Mass of plasmid DNA needed (ng) |Final concentration (ng/ul) of DNA |

|100000000 |1.167 |0.233 |

|10000000 |0.1167 |0.0233 |

|1000000 |0.01167 |0.00233 |

|100000 |0.001167 |0.000233 |

|10000 |0.0001167 |0.0000233 |

|1000 |0.00001167 |0.00000233 |

|100 |0.000001167 |0.000000233 |

|10 |0.0000001167 |0.0000000233 |

|1 |0.00000001167 |0.00000000233 |

Foamy vector genomic DNA isolation (5 ul)

This protocol isolates vector DNA from virions. This is the cDNA molecule and is mostly a double-stranded linear genome.

1. Rapidly thaw 5 ul of FV vector stock at 37C in H2O bath.

2. Add 500 ul of TE to stock in a 1.5 ml microfuge tube.

3. Add equal volume of phenol/chloroform, mix well by inverting tube, and spin for 1 minute at 13,200 rpm in a microfuge.

4. Collect aqueous phase and transfer to a new microfuge tube.

5. Repeat steps 3 and 4.

6. Add equal volume of 24:1 (v/v) chloroform-isoamyl alcohol, invert several times, and spin for 1 minute at 13,200 rpm in a microfuge.

7. Collect aqueous phase and transfer to a new microfuge tube.

8. Add 2 ul Pellet Paint Carrier Non-Fluorescent (Novagen) or equivalent carrier.

9. Precipitate DNA by adding 0.1 volume of 3 M sodium acetate, pH 5.2, to the aqueous phase and 2 volumes of 100% ethanol. Mix by inverting. Sit at room temp for 2 minutes.

10. Recover DNA by centrifugation at 13,200 rpm in a microfuge for 5 minutes.

11. Carefully remove supernatant and then wash pellet with 750 ul of 70% ethanol and spin for 5 minutes at 13,200 rpm.

12. Carefully remove ethanol and air dry pellet for 10 minutes at room temperature.

13. Resuspend DNA in 50 ul of sterile, PCR-clean TE, pH 8.0.

Foamy vector genomic DNA DpnI digestion

This step is used to digest contaminating, methylated plasmid DNA in the vector stock. Dpn I will not digest the unmethylated FV vector genome, so this will be the PCR template.

1. Take 12 ul of foamy vector genomic DNA, add 2 ul of NEB Buffer#4 and 1 ul of DpnI and 4 ul dH2O and 1 ul BSA (10X NEB). Digest at 37C for 2 hours.

2. Add 100 ul of TE to the digestion solution. Each ul of the digest represents 0.01 ul of original vector stock. We will use 0.05 ul of original vector stock in each PCR reaction.

Foamy vector genomic DNA isolation (50 ul)

This protocol isolates DNA from 10 times the amount of vector as the protocol on the previous page. It includes more extractions so is not as convenient. The advantage is that one gets enough DNA to Southern blots and confirm the qPCR results and characterize the vector genome forms. This is useful sometimes to more carefully analyze a stock and identify possible unexpected genome structures.

1. Rapidly thaw 50 ul of FV vector stock at 37C in H2O bath.

2. Add 500 ul of TE to stock in a 1.5 ml microfuge tube.

3. Add an equal volume of phenol, mix well by inverting, and spin for 1 minute at 13,200 rpm in a microfuge.

4. Collect aqueous phase and transfer to a new microfuge tube.

5. Repeat phenol extraction (steps 3 and 4).

6. Add equal volume of phenol/chloroform, mix well by inverting tube, and spin for 1 minute at 13,200 rpm in a microfuge.

7. Collect aqueous phase and transfer to a new microfuge tube.

8. Repeat phenol/chloroform extraction (steps 6 and 7).

9. Add equal volume of 24:1 (v/v) chloroform-isoamyl alcohol, invert several times, and spin for 1 minute at 13,200 rpm in a microfuge.

10. Collect aqueous phase and transfer to a new microfuge tube.

11. Add 2 ul Pellet Paint Carrier Non-Fluorescent (Novagen) or equivalent carrier.

12. Precipitate DNA by adding 0.1 volume of 3 M sodium acetate, pH 5.2, to the aqueous phase and 2 volumes of 100% ethanol. Mix by inverting. Sit at room temp for 2 minutes.

13. Recover DNA by centrifugation at 13,200 rpm in a microfuge for 5 minutes.

14. Carefully remove supernatant and then wash pellet with 750 ul of 70% ethanol and spin for 5 minutes at 13,200 rpm.

15. Carefully remove ethanol and air dry pellet for 10 minutes at room temperature.

16. Resuspend DNA in 50 ul of sterile, PCR-clean TE, pH 8.0.

Foamy vector genomic DNA DpnI digestion

This step is used to digest contaminating, methylated plasmid DNA in the vector stock. Dpn I will not digest the unmethylated FV vector genome, so this will be the PCR template.

1. Take 12 ul of foamy vector genomic DNA, add 2 ul of NEB Buffer#4 and 1 ul of DpnI and 4 ul dH2O and 1 ul BSA (10X NEB). Digest at 37C for 2 hours.

2. Add 1180 ul of TE to the digestion solution. Each ul of the digest represents 0.01 ul of original vector stock. We will use 0.05 ul of original vector stock in each PCR reaction.

qPCR reaction

The qPCR reaction amplifies a region of the provirus that is not present in the original vector plasmid used to make the stock (the plasmid contains a CMV-LTR fusion promoter at its 5’ end that is replaced by a true FV LTR in the provirus). This increases the specificity of the reaction.

1. Reconstitute the primers in sterile, nuclease-free dH2O to a working stock concentration of 50 uM. Aliquot 30 ul/tube x 10 tubes. Store at -20C.

2. Dilute TaqMan probe (100 uM) to a working stock concentration of 10 uM using TE. Aliquot 30 ul/tube x 10 tubes. Store at -20C in the dark.

3. Before making reaction mix, turn StepOnePlus™ Real time PCR machine on. Set up a file containing information about your plate (detector, task, sample name, standard copy numbers, and sample distribution on the plate). Refer to manual from Applied Biosystems. Change reaction volume to 25 ul (default is 20 ul) and PCR cycle number to 45 (default is 40). Save the file.

4. Prepare a PCR mix cocktail with primer concentrations of 900 nM and TaqMan probe concentration of 250 nM. Determine the number of reactions that the experiment will require and add an additional 10% volume than what is needed.

For example, if you need to do 25 reactions, make a master mix for 28 reactions (see table below). Combine 350 ul of 2X TaqMan( Gene Expression Master Mix, 12.6 ul of each primer, 17.5 ul of probe and 167.3 ul of dH2O in a microfuge tube. Vortex well.

|Reagent |Stock Concentration|Volume (ul) in single |Volume (ul) needed for mix|Final |

| | |reaction |(x28) |concentration |

|TaqMan Universal PCR |2X |12.5 |350 |1X |

|Master Mix | | | | |

|Forward primer |50 uM |0.45 |12.6 |900 nM |

|Reverse primer |50 uM |0.45 |12.6 |900 nM |

|TaqMan probe |10 uM |0.625 |17.5 |250 nM |

|Sterile dH2O |N/A |5.975 |167.3 |N/A |

|Total | |20 |560 | |

5. Into each designated well of the reaction plate (according to your plate design previously), add 20 ul of the PCR mix cocktail. Every plate must include negative control using dH2O instead of sample.

6. Add 5 ul of the pACT5FVMF(f) serial dilutions or DpnI-digested FV vector DNA to each reaction. Standard plasmid and FV genomic DNA samples should be duplicated. Cover the plate with Optical Adhesive Film (Applied Biosystems).

7. Spin down the liquid at 1200 rpm, at 4C for 2 min (we use Allegra™ X-12R centrifuge from Beckman Coulter).

8. Put plate into StepOnePlus Real time PCR machine and start run program. (2 min at 50C, 10 min at 95C, 15 sec at 95C, 1 min @ 60C).

9. Analyze data

pACT5FVMF(f)

LOCUS pACT5FVMF( 10644 bp DNA CIRCULAR SYN 03-APR-2007

DEFINITION Ligation of inverted MLV_LTR_B*Xho1Xma1 into ACT5DeltaPhiPFpv(f)* AgeIXho1

ACCESSION pACT5FVMF(

KEYWORDS .

SOURCE Unknown.

ORGANISM Unknown

Unclassified.

REFERENCE 1 (bases 1 to 10644)

AUTHORS Self

JOURNAL Notebook

COMMENT SECID/File created by SciEd Central, Scientific & Educational Software

FEATURES Location/Qualifiers

misc_feature 1..250

/gene="TRE"

/product="Tet response element from pTRE-Tight (Clontech)"

misc_feature 256..315

/gene="Pcmv"

/product="minimal CMV promoter from pTRE-Tight (Clontech)"

misc_feature 316..347

/gene="MCS"

misc_feature 348..932

/gene="IRES"

/product="IRES from pIRES2-EGFP (Clontech)the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV)"

CDS 924..2588

/gene="luc"

/product="firefly luciferase gene"

misc_feature 2648..2691

/gene="SpA"

/product="synthetic polyadenylation site based on rabbit beta-globin gene; Xba1 and Pci1 ends with Mlu1 cloning site"

misc_feature 2756..3355

/gene="ColE1"

CDS complement (3517..4377)

/gene="amp"

misc_feature 4818..5001

/gene="deltaU3"

misc_feature 5002..5360

/gene="R-U5"

misc_feature 5468..5639

/gene="gag'"

misc_feature 5640..7539

/gene="Polseq'"

misc_feature 7486..7952

/gene="env'"

misc_feature 7995..8469

/gene="MLV'"

/product="MLV proviral LTR"

CDS 8482..9201

/gene="EGFP"

misc_feature 9288..9830

/gene="3-deltaU3-LTR"

misc_feature 9849..10644

/gene="spacer"

/product="795 bp PCR product of HPRT intron B(2) cut with Sal1"

BASE COUNT 3059 a 2356 c 2353 g 2876 t

ORIGIN

1 CGAGTTTACT CCCTATCAGT GATAGAGAAC GTATGTCGAG TTTACTCCCT ATCAGTGATA

61 GAGAACGATG TCGAGTTTAC TCCCTATCAG TGATAGAGAA CGTATGTCGA GTTTACTCCC

121 TATCAGTGAT AGAGAACGTA TGTCGAGTTT ACTCCCTATC AGTGATAGAG AACGTATGTC

181 GAGTTTATCC CTATCAGTGA TAGAGAACGT ATGTCGAGTT TACTCCCTAT CAGTGATAGA

241 GAACGTATGT CGAGGTAGGC GTGTACGGTG GGAGGCCTAT ATAAGCAGAG CTCGTTTAGT

301 GAACCGTCAG ATCGCCTGGA GAATTCGAGC TCGGTACCCG GGGATCCGCC CCTCTCCCTC

361 CCCCCCCCCT AACGTTACTG GCCGAAGCCG CTTGGAATAA GGCCGGTGTG CGTTTGTCTA

421 TATGTTATTT TCCACCATAT TGCCGTCTTT TGGCAATGTG AGGGCCCGGA AACCTGGCCC

481 TGTCTTCTTG ACGAGCATTC CTAGGGGTCT TTCCCCTCTC GCCAAAGGAA TGCAAGGTCT

541 GTTGAATGTC GTGAAGGAAG CAGTTCCTCT GGAAGCTTCT TGAAGACAAA CAACGTCTGT

601 AGCGACCCTT TGCAGGCAGC GGAACCCCCC ACCTGGCGAC AGGTGCCTCT GCGGCCAAAA

661 GCCACGTGTA TAAGATACAC CTGCAAAGGC GGCACAACCC CAGTGCCACG TTGTGAGTTG

721 GATAGTTGTG GAAAGAGTCA AATGGCTCTC CTCAAGCGTA TTCAACAAGG GGCTGAAGGA

781 TGCCCAGAAG GTACCCCATT GTATGGGATC TGATCTGGGG CCTCGGTGCA CATGCTTTAC

841 ATGTGTTTAG TCGAGGTTAA AAAAACGTCT AGGCCCCCCG AACCACGGGG ACGTGGTTTT

901 CCTTTGAAAA ACACGATGAT AATATGGCCA CAACCATGGA AGACGCCAAA AACATAAAGA

961 AAGGCCCGGC GCCATTCTAT CCTCTAGAGG ATGGAACCGC TGGAGAGCAA CTGCATAAGG

1021 CTATGAAGAG ATACGCCCTG GTTCCTGGAA CAATTGCTTT TACAGATGCA CATATCGAGG

1081 TGAACATCAC GTACGCGGAA TACTTCGAAA TGTCCGTTCG GTTGGCAGAA GCTATGAAAC

1141 GATATGGGCT GAATACAAAT CACAGAATCG TCGTATGCAG TGAAAACTCT CTTCAATTCT

1201 TTATGCCGGT GTTGGGCGCG TTATTTATCG GAGTTGCAGT TGCGCCCGCG AACGACATTT

1261 ATAATGAACG TGAATTGCTC AACAGTATGA ACATTTCGCA GCCTACCGTA GTGTTTGTTT

1321 CCAAAAAGGG GTTGCAAAAA ATTTTGAACG TGCAAAAAAA ATTACCAATA ATCCAGAAAA

1381 TTATTATCAT GGATTCTAAA ACGGATTACC AGGGATTTCA GTCGATGTAC ACGTTCGTCA

1441 CATCTCATCT ACCTCCCGGT TTTAATGAAT ACGATTTTGT ACCAGAGTCC TTTGATCGTG

1501 ACAAAACAAT TGCACTGATA ATGAATTCCT CTGGATCTAC TGGGTTACCT AAGGGTGTGG

1561 CCCTTCCGCA TAGAACTGCC TGCGTCAGAT TCTCGCATGC CAGAGATCCT ATTTTTGGCA

1621 ATCAAATCAT TCCGGATACT GCGATTTTAA GTGTTGTTCC ATTCCATCAC GGTTTTGGAA

1681 TGTTTACTAC ACTCGGATAT TTGATATGTG GATTTCGAGT CGTCTTAATG TATAGATTTG

1741 AAGAAGAGCT GTTTTTACGA TCCCTTCAGG ATTACAAAAT TCAAAGTGCG TTGCTAGTAC

1801 CAACCCTATT TTCATTCTTC GCCAAAAGCA CTCTGATTGA CAAATACGAT TTATCTAATT

1861 TACACGAAAT TGCTTCTGGG GGCGCACCTC TTTCGAAAGA AGTCGGGGAA GCGGTTGCAA

1921 AACGCTTCCA TCTTCCAGGG ATACGACAAG GATATGGGCT CACTGAGACT ACATCAGCTA

1981 TTCTGATTAC ACCCGAGGGG GATGATAAAC CGGGCGCGGT CGGTAAAGTT GTTCCATTTT

2041 TTGAAGCGAA GGTTGTGGAT CTGGATACCG GGAAAACGCT GGGCGTTAAT CAGAGAGGCG

2101 AATTATGTGT CAGAGGACCT ATGATTATGT CCGGTTATGT AAACAATCCG GAAGCGACCA

2161 ACGCCTTGAT TGACAAGGAT GGATGGCTAC ATTCTGGAGA CATAGCTTAC TGGGACGAAG

2221 ACGAACACTT CTTCATAGTT GACCGCTTGA AGTCTTTAAT TAAATACAAA GGATATCAGG

2281 TGGCCCCCGC TGAATTGGAA TCGATATTGT TACAACACCC CAACATCTTC GACGCGGGCG

2341 TGGCAGGTCT TCCCGACGAT GACGCCGGTG AACTTCCCGC CGCCGTTGTT GTTTTGGAGC

2401 ACGGAAAGAC GATGACGGAA AAAGAGATCG TGGATTACGT CGCCAGTCAA GTAACAACCG

2461 CGAAAAAGTT GCGCGGAGGA GTTGTGTTTG TGGACGAAGT ACCGAAAGGT CTTACCGGAA

2521 AACTCGACGC AAGAAAAATC AGAGAGATCC TCATAAAGGC CAAGAAGGGC GGAAAGTCCA

2581 AATTGTAAAA TGTAACTGTA TTCAGCGATG ACGAAATTCT TAGCTATTGT AATGACTCTA

2641 GACGCGTAAT AAAAGATCCT TATTTTCATT GGATCTGTGT GTTGGTTTTT TGTGTGACAT

2701 GTGAGCAAAA GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT

2761 CCATAGGCTC CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG

2821 AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC

2881 TCCTGTTCCG ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT

2941 GGCGCTTTCT CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA

3001 GCTGGGCTGT GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA

3061 TCGTCTTGAG TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA

3121 CAGGATTAGC AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA

3181 CTACGGCTAC ACTAGAAGGA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT

3241 CGGAAAAAGA GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT

3301 TTTTGTTTGC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT

3361 CTTTTCTACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT

3421 GAGATTATCA AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC

3481 AATCTAAAGT ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC

3541 ACCTATCTCA GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA

3601 GATAACTACG ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA

3661 CCCACGCTCA CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG

3721 CAGAAGTGGT CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC

3781 TAGAGTAAGT AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT

3841 CGTGGTGTCA CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG

3901 GCGAGTTACA TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT

3961 CGTTGTCAGA AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA

4021 TTCTCTTACT GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA

4081 GTCATTCTGA GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA

4141 TAATACCGCG CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG

4201 GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC

4261 ACCCAACTGA TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG

4321 AAGGCAAAAT GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT

4381 CTTCCTTTTT CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT

4441 ATTTGAATGT ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT

4501 GCCACCTAAA TTGTAAGCGT TAATATTTTG TTAAAATTCG CGTTAAATTT TTGTTAAATC

4561 AGCTCATTTT TTAACCAATA GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG

4621 ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG

4681 GACTCCAACG TCAAAGGGCG AAAAACCGTC TATCAGGGCG ATGGCCCACT ACATCAAGCT

4741 TATCGATACC GTCGACCGTC AATGGGTGGA GTATTTACGG TAAACTGCCC ACTTGGCAGT

4801 ACATCAAGTG TATCATATGT GGTGGAATGT CACTAGAAAC CAGGGAAAAC AAGGAGGAGA

4861 GTATTACAGG GAAGGAGGTG AAGAACCTCA TTACCCAAAT ACTCCTGCTC CTCATAGACG

4921 TACCTGGGAT GAGAGACACA AGGTTCTTAA ATTGTCCTCA TTCGCTACTC CCTCTGACAT

4981 CCAACGCTGG GCTACTAACT CTAGATTGTA CGGGAGCTCT CTTCACTACT CGCTGCGTCG

5041 AGAGTGTACG AGACTCTCCA GGTTTGGTAA GAAATATTTT ATATTGTTAT AATGTTACTA

5101 TGATCCATTA ACACTCTGCT TATAGATTGT AAGGGTGATT GCAATGCTTT CTGCATAAAA

5161 CTTTGGTTTT CTTGTTAATC AATAAACCGA CTTGATTCGA GAACCTACTC ATATATTATT

5221 GTCTCTTTTA TACTTTATTA AGTAAAAGGA TTTGTATATT AGCCTTGCTA AGGGAGACAT

5281 CTAGTGATAT AAGTGTGAAC TACACTTATC TTAAATGATG TAACTCCTTA GGATAATCAA

5341 TATACAAAAT TCCATGACAA TTGGCGCCCA ACGTGGGGCT CGAATATAAG TCGGGTTTAT

5401 TTGTAAATTA TCCCTAGGGA CCTCCGAGCA AGCGAGCATA GCGGGAGGCA TATAAAAGCC

5461 AATAGACAAT GGCTAGCAGG AAGTAATGTT GAAGAATATG AACTTGATGT TGAAGCTCTG

5521 GTTGTAATTT TAAGAGATAG AAATATACCA AGAAATCCTT TACATGGAGA AGTTATAGGT

5581 CTTCGCCTTA CTGAAGGATG GTGGGGACAA ATTGAGAGAT TTCAGATGGT ACGTTGATCT

5641 AAGGCTATGG ATTTGGCCAT GGGACAAGAA ATATTAGTTT ATAGTCCCAT TGTATCTATG

5701 ACTAAAATAC AAAAAACTCC ACTACCAGAA AGAAAAGCTT TACCCATTAG ATGGATAACA

5761 TGGATGACTT ATTTAGAAGA TCCAAGAATC CAATTTCATT ATGATAAAAC CTTACCAGAA

5821 CTTAAGCATA TTCCAGATGT ATATACATCT AGTCAGTCTC CTGTTAAACA TCCTTCTCAA

5881 TATGAAGGAG TGTTTTATAC TGATGGCTCG GCCATCAAAA GTCCTGATCC TACAAAAAGC

5941 AATAATGCTG GCATGGGAAT AGTACATGCC ACATACAAAC CTGAATATCA AGTTTTGAAT

6001 CAATGGTCAA TACCACTAGG TAATCATACT GCTCAGATGG CTGAAATAGC TGCAGTTGAA

6061 TTTGCCTGTA AAAAAGCTTT AAAAATACCT GGTCCTGTAT TAGTTATAAC TGATAGTTTC

6121 TATGTAGCAG AAAGTGCTAA TAAAGAATTA CCATACTGGA AATCTAATGG GTTTGTTAAT

6181 AATAAGAAAA AGCCTCTTAA ACATATCTCC AAATGGAAAT CTATTGCTGA GTGTTTATCT

6241 ATGAAACCAG ACATTACTAT TCAACATGAA AAAGGCATCA GCCTACAAAT ACCAGTATTC

6301 ATACTGAAAG GCAATGCCCT AGCAGATAAG CTTGCCACCC AAGGAAGTTA TGTGGTTAAT

6361 TGTAATACCA AAAAACCAAA CCTGGATGCA GAGTTGGATC AATTATTACA GGGTCATTAT

6421 ATAAAAGGAT ATCCCAAACA ATATACATAT TTTTTAGAAG ATGGCAAAGT AAAAGTTTCC

6481 AGACCTGAAG GGGTTAAAAT TATTCCCCCT CAGTCAGACA GACAAAAAAT TGTGCTTCAA

6541 GCCCACAATT TGGCTCACAC CGGACGTGAA GCCACTCTTT TAAAAATTGC CAACCTTTAT

6601 TGGTGGCCAA ATATGAGAAA GGATGTGGTT AAACAACTAG GACGCTGTCA ACAGTGTTTA

6661 ATCACAAATG CTTCCAACAA AGCCTCTGGT CCTATTCTAA GACCAGATAG GCCTCAAAAA

6721 CCTTTTGATA AATTCTTTAT TGACTATATT GGACCTTTGC CACCTTCACA GGGATACCTA

6781 TATGTATTAG TAGTTGTTGA TGGAATGACA GGATTCACTT GGTTATACCC CACTAAGGCT

6841 CCTTCTACTA GCGCAACTGT TAAATCTCTC AATGTACTCA CTAGTATTGC AATTCCAAAG

6901 GTGATTCACT CTGATCAAGG TGCAGCATTC ACTTCTTCAA CCTTTGCTGA ATGGGCAAAG

6961 GAAAGAGGTA TACATTTGGA ATTCAGTACT CCTTATCACC CCCAAAGTGG TAGTAAGGTG

7021 GAAAGGAAAA ATAGTGATAT AAAACGACTT TTAACTAAAC TGCTAGTAGG AAGACCCACA

7081 AAGTGGTATG ACCTATTGCC TGTTGTACAA CTTGCTTTAA ACAACACCTA TAGCCCTGTA

7141 TTAAAATATA CTCCACATCA ACTCTTATTT GGTATAGATT CAAATACTCC ATTTGCAAAT

7201 CAAGATACAC TTGACTTGAC CAGAGAAGAA GAACTTTCTC TTTTACAGGA AATTCGTACT

7261 TCTTTATACC ATCCATCCAC CCCTCCAGCC TCCTCTCGTT CCTGGTCTCC TGTTGTTGGC

7321 CAATTGGTCC AGGAGAGGGT GGCTAGGCCT GCTTCTTTGA GACCTCGTTG GCATAAACCG

7381 TCTACTGTAC TTAAGGTGTT GAATCCAAGG ACTGTTGTTA TTTTGGACCA TCTTGGCAAC

7441 AACAGAACTG TAAGTATAGA TAATTTAAAA CCTACTTCTC ATCAGAATGG CACCACCAAT

7501 GACACTGCAA CAATGGATCA TTTGGAAAAA AATGAATAAA GCGCATGAGG CACTTCAAAA

7561 TACAACAACT GTGACTGAAC AGCAGAAGGA ACAAATTATA CTGGACATTC AAAATGAAGA

7621 AGTACAACCA ACTAGGAGAG ATAAATTTAG ATATCTGCTT TATACTTGTT GTGCTACTAG

7681 CTCAAGAGTA TTGGCCTGGA TGTTTTTAGT TTGTATATTG TTAATCATTG TTTTGGTTTC

7741 ATGCTTTGTG ACTATATCCA GAATACAATG GAATAAGGAT ATTCAGGTAT TAGGACCTGT

7801 AATAGACTGG AATGTTACTC AAAGAGCTGT TTATCAACCC TTACAGACTA GAAGGATTGC

7861 ACGTTCCCTT AGAATGCAGC ATCCTGTTCC AAAATATGTG GAGGTAAATA TGACTAGTAT

7921 TCCACAAGGT GTATACTATG AACCCCATCC GGCCTCGACG GTATCGATAA GCTTGATAAC

7981 CTCGAGGTCG ACGTTGAAAG ACCCCACCTG TAGGTTTGGC AAGCTAGCTT AAGTAACGCC

8041 ATTTTGCAAG GCATGGAAAA ATACATAACT GAGAATAGAG AAGTTCAGAT CAAGGTCAGG

8101 AACAGATGGA ACAGCTGAAT ATGGGCCAAA CAGGATATCT GTGGTAAGCA GTTCCTGCCC

8161 CGGCTCAGGG CCAAGAACAG ATGGAACAGC TGAATATGGG CCAAACAGGA TATCTGTGGT

8221 AAGCAGTTCC TGCCCCGGCT CAGGGCCAAG AACAGATGGT CCCCAGATGC GGTCCAGCCC

8281 TCAGCAGTTT CTAGAGAACC ATCAGATGTT TCCAGGGTGC CCCAAGGACC TGAAATGACC

8341 CTGTGCCTTA TTTGAACTAA CCAATCAGTT CGCTTCTCGC TTCTGTTCGC GCGCTTCTGC

8401 TCCCCGAGCT CAATAAAAGA GCCCACAACC CCTCACTCGG GGCGCCAGTC CTCCGATTGA

8461 CTGAGTCGCC CGGTCGCCAC CATGGTGAGC AAGGGCGAGG AGCTGTTCAC CGGGGTGGTG

8521 CCCATCCTGG TCGAGCTGGA CGGCGACGTA AACGGCCACA AGTTCAGCGT GTCCGGCGAG

8581 GGCGAGGGCG ATGCCACCTA CGGCAAGCTG ACCCTGAAGT TCATCTGCAC CACCGGCAAG

8641 CTGCCCGTGC CCTGGCCCAC CCTCGTGACC ACCCTGACCT ACGGCGTGCA GTGCTTCAGC

8701 CGCTACCCCG ACCACATGAA GCAGCACGAC TTCTTCAAGT CCGCCATGCC CGAAGGCTAC

8761 GTCCAGGAGC GCACCATCTT CTTCAAGGAC GACGGCAACT ACAAGACCCG CGCCGAGGTG

8821 AAGTTCGAGG GCGACACCCT GGTGAACCGC ATCGAGCTGA AGGGCATCGA CTTCAAGGAG

8881 GACGGCAACA TCCTGGGGCA CAAGCTGGAG TACAACTACA ACAGCCACAA CGTCTATATC

8941 ATGGCCGACA AGCAGAAGAA CGGCATCAAG GTGAACTTCA AGATCCGCCA CAACATCGAG

9001 GACGGCAGCG TGCAGCTCGC CGACCACTAC CAGCAGAACA CCCCCATCGG CGACGGCCCC

9061 GTGCTGCTGC CCGACAACCA CTACCTGAGC ACCCAGTCCG CCCTGAGCAA AGACCCCAAC

9121 GAGAAGCGCG ATCACATGGT CCTGCTGGAG TTCGTGACCG CCGCCGGGAT CACTCTCGGC

9181 ATGGACGAGC TGTACAAGTA AAGCGGCCGC GACTCTAGAT CAGGCGCGCC GTTACCAAGC

9241 AGCTATGGAA GCTTATGGAC CTCAGAGAGG AAGTAACGAG GAGAGGGTGT GGTGGAATGT

9301 CACTAGAAAC CAGGGAAAAC AAGGAGGAGA GTATTACAGG GAAGGAGGTG AAGAACCTCA

9361 TTACCCAAAT ACTCCTGCTC CTCATAGACG TACCTGGGAT GAGAGACACA AGGTTCTTAA

9421 ATTGTCCTCA TTCGCTACTC CCTCTGACAT CCAACGCTGG GCTACTAACT CTAGATTGTA

9481 CGGGAGCTCT CTTCACTACT CGCTGCGTCG AGAGTGTACG AGACTCTCCA GGTTTGGTAA

9541 GAAATATTTT ATATTGTTAT AATGTTACTA TGATCCATTA ACACTCTGCT TATAGATTGT

9601 AAGGGTGATT GCAATGCTTT CTGCATAAAA CTTTGGTTTT CTTGTTAATC AATAAACCGA

9661 CTTGATTCGA GAACCTACTC ATATATTATT GTCTCTTTTA TACTTTATTA AGTAAAAGGA

9721 TTTGTATATT AGCCTTGCTA AGGGAGACAT CTAGTGATAT AAGTGTGAAC TACACTTATC

9781 TTAAATGATG TAACTCCTTA GGATAATCAA TATACAAAAT TCCATGACAA TTGGCGATAC

9841 CGTCGACCTC GACGGACTGG GGCTGAATCA CATGAAGGCA AGGTCAGATT TTTATTATTA

9901 TGCACATCTA GCTTGAAAAT TTTCTGTTAA GTCAATTACA GTGAAAAACC TTACCTGGTA

9961 TTGAATGCTT GCATTGTATG TCTGGCTATT CTGTGTTTTT ATTTTAAAAT TATAATATCA

10021 AAATATTTGT GTTATAAAAT ATTCTAACTA TGGAGGCCAT AAACAAGAAG ACTAAAGTTC

10081 TCTCCTTTCA GCCTTCTGTA CACATTTCTT CTCAAGCACT GGCCTATGCA TGTATACTAT

10141 ATGCAAAAGT ACATATATAC ATTTATATTT TAACGTATGA GTATAGTTTT AAATGTTATT

10201 GGACACTTTT AATATTAGTG TGTCTAGAGC TATCTAATAT ATTTTAAAGG TTGCATAGCA

10261 TTCTGTCTTA TGGAGATACC ATAACTGATT TAACCAGTCC ACTATTGATA GACACTATTT

10321 TGTTCTTACC GACTGTACTA GAAGAAACAT TCTTTTACAT GTTTGGTACT TGTTCAGCTT

10381 TATTCAAGTG GAATTTCTGG GTCAAGGGGA AAGAGTTTAT TGAATATTTT GGTATTGCCA

10441 AATTTTCCTC TAAGAAGTTG AATCATTTTA TACTCCTGAT GTTATATGAG AGTACCTTTC

10501 TCTTCACAAT TTGTCTCTTT TTTTTTTTTT TTTGAGACAA GGTCTCTGTT GCCCAGGCTG

10561 GGGTGCAGTG CAGCAGAATG ATCACAGTTC ACTGCAGTCT CAACCTCCTG GGTTCAAGCG

10621 ATCCTTCCAC CTCAGCCTCC TGGT

//

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download