Sequencing SPR1 gene

The SPR1 gene was taken by digesting SPR1bBluescript plasmid and replaced to T7-Luciferase plasmid in the position of the enzyme (mentioned). The gene was amplified by specific PCR using SPR1BAMF1 and SPR1SACSTOPR1 primers, digested with BamH1 and Sac1, purified and ligated into BamH1/Sac1 digested T7-Luc plasmid. The purified recombinant vector was amplified in Highly competent E. coli NEB 5-alpha cells. The purified plasmid from the cells were used as template for sequencing reaction using 3.2 pmol/microliter T7 promoter primer and 5 pmol/microliter SPR1SACSTOPR1primer in separate reactions to get two (forward and reverse sequencing) results to compare.

Group5 T7 9-20-11.ab1 interpretation:

Sequence obtained:

NNNNNNNNNNNNNNNNNNNNNNNNN AGGGCCCGGATCCATGGGTCGTGGAAACAGCTGTGGTGGAGGTCAAAGCTNNNNNNNNNNTCTCTTTGGTGGTGACGCTCCTGCTCCTAAGCCAGTTCCAGCTCCTCGTCCCGCTCCTACTGAGTCTAACAACGGACCTGCACCACCAGTAACAGCTGTGACTGCAACCGCACTCACGACTGCTACTACTTCTGTTGAGCCTGCAGAGCTTAACAAGCAGATTCCTGCTGGTATCAAAACTCCTGTTAACAACTATGCCAGAGCTGAAGGACAGAACACCGGCAACTTCCTCACTGACCGTCCTTCGACCAAAGTTCACGCAGCTCCGGGAGGAGGATCATCCTTGGATTATCTCTTCACTGGTGGCAAGTAAGAGCTCCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCGAATTGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGNGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCNAATGCAGGAGTCGCATANNNANAGCGTCNAATGNNCACTCTCAGTACNNNCTGCTCTGANGCCGCATANTNNCCANNCCNNNNNCCNNNNNCCNNTNACGCGCCNGANGGNNTGNNNGNNNNNATCNNTNNNANNANNTNNNNNNNGNNNNNNNNTGCATGNNNNNNGTTNNCNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNTNNGNNNNNCNNNNNANNNNNNNNNNNAN

Sequence Analysis using FinchTV image:

First 25 Ntds are not assigned by the sequencer = Ntds in T7 Luc Backbone vector

BamH1 restriction site from 33 – 38 Ntds

SPR1 gene starts from 39^th Ntd

Ntds from 75 – 85: No Ntd was assigned.

Based on the peaks, the Ntds may be CATTGGATTA

The sequence exactly matches in the known SPR1 gene sequence (highlighted)

ATGGGTCGTGGAAACAGCTGTGGTGGAGGTCAAAGCTCATTGGATTATCTCTTTGGTGGTGACGCTCCTGCTCCTAAGCCAGTTCCAGCTCCTCGTCCCGCTCCTACTGAGTCTAACAACGGACCTGCACCACCAGTAACAGCTGTGACTGCAACCGCACTCACGACTGCTACTACTTCTGTTGAGCCTGCAGAGCTTAACAAGCAGATTCCTGCTGGTATCAAAACTCCTGTTAACAACTATGCCAGAGCTGAAGGACAGAACACCGGCAACTTCCTCACTGACCGTCCTTCGACCAAAGTTCACGCAGCTCCGGGAGGAGGATCATCCTTGGATTATCTCTTCACTGGTGGCAAGTAA

Poly A residues were observed from 407 – 436^th positions with short height peaks.

Just before the polyA residues, stop sequence (TAA, 396-398th) and SacI restriction site (399 – 404^th) was found.

Rest of the Ntds must be from vector backbone.

The “Noise” increased after polyA sequence.

The height of the peaks seemed to be vague after 541th Ntd.

After 681th Ntd, the sequence identification was largely inconclusive limiting the size (length) of the sequence. (Actually, the sequencer can sequence good to around 800 Ntds. The lower value might be due to contamination during plasmid preparation. This indication was also observed on the gel electrophoresis result of the digested recombinant construct.)

97% similarities of the sequenced SPR1 gene were observed for all three entries in the NCBI Blasting as: Identifying the unknown Ntds (as n in the query sequence) will completely match the sequences. Therefore, the sequenced result was good.

>ref|NM_001161019.1| Arabidopsis thaliana spiral1 (SPR1) mRNA, complete cds

Length=854

GENE ID: 814896 SPR1 | spiral1 [Arabidopsis thaliana]

(10 or fewer PubMed links)

Score = 628 bits (340), Expect = 2e-178

Identities = 350/360 (97%), Gaps = 0/360 (0%)

Strand=Plus/Plus

Query 14 ATGGGTCGTGGAAACAGCTGTGGTGGAGGTCAAAGCTnnnnnnnnnnTCTCTTTGGTGGT 73

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

Sbjct 290 ATGGGTCGTGGAAACAGCTGTGGTGGAGGTCAAAGCTCATTGGATTATCTCTTTGGTGGT 349

Query 74 GACGCTCCTGCTCCTAAGCCAGTTCCAGCTCCTCGTCCCGCTCCTACTGAGTCTAACAAC 133

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

Sbjct 350 GACGCTCCTGCTCCTAAGCCAGTTCCAGCTCCTCGTCCCGCTCCTACTGAGTCTAACAAC 409

Query 134 GGACCTGCACCACCAGTAACAGCTGTGACTGCAACCGCACTCACGACTGCTACTACTTCT 193

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

Sbjct 410 GGACCTGCACCACCAGTAACAGCTGTGACTGCAACCGCACTCACGACTGCTACTACTTCT 469

Query 194 GTTGAGCCTGCAGAGCTTAACAAGCAGATTCCTGCTGGTATCAAAACTCCTGTTAACAAC 253

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

Sbjct 470 GTTGAGCCTGCAGAGCTTAACAAGCAGATTCCTGCTGGTATCAAAACTCCTGTTAACAAC 529

Query 254 TATGCCAGAGCTGAAGGACAGAACACCGGCAACTTCCTCACTGACCGTCCTTCGACCAAA 313

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

Sbjct 530 TATGCCAGAGCTGAAGGACAGAACACCGGCAACTTCCTCACTGACCGTCCTTCGACCAAA 589

Query 314 GTTCACGCAGCTCCGGGAGGAGGATCATCCTTGGATTATCTCTTCACTGGTGGCAAGTAA 373

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

Sbjct 590 GTTCACGCAGCTCCGGGAGGAGGATCATCCTTGGATTATCTCTTCACTGGTGGCAAGTAA 649

>ref|NM_126416.2| Arabidopsis thaliana spiral1 (SPR1) mRNA, complete cds

Length=659

GENE ID: 814896 SPR1 | spiral1 [Arabidopsis thaliana]

(10 or fewer PubMed links)

Score = 628 bits (340), Expect = 2e-178

Identities = 350/360 (97%), Gaps = 0/360 (0%)

Strand=Plus/Plus

Query 14 ATGGGTCGTGGAAACAGCTGTGGTGGAGGTCAAAGCTnnnnnnnnnnTCTCTTTGGTGGT 73

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

Sbjct 95 ATGGGTCGTGGAAACAGCTGTGGTGGAGGTCAAAGCTCATTGGATTATCTCTTTGGTGGT 154

Query 74 GACGCTCCTGCTCCTAAGCCAGTTCCAGCTCCTCGTCCCGCTCCTACTGAGTCTAACAAC 133

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

Sbjct 155 GACGCTCCTGCTCCTAAGCCAGTTCCAGCTCCTCGTCCCGCTCCTACTGAGTCTAACAAC 214

Query 134 GGACCTGCACCACCAGTAACAGCTGTGACTGCAACCGCACTCACGACTGCTACTACTTCT 193

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

Sbjct 215 GGACCTGCACCACCAGTAACAGCTGTGACTGCAACCGCACTCACGACTGCTACTACTTCT 274

Query 194 GTTGAGCCTGCAGAGCTTAACAAGCAGATTCCTGCTGGTATCAAAACTCCTGTTAACAAC 253

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

Sbjct 275 GTTGAGCCTGCAGAGCTTAACAAGCAGATTCCTGCTGGTATCAAAACTCCTGTTAACAAC 334

Query 254 TATGCCAGAGCTGAAGGACAGAACACCGGCAACTTCCTCACTGACCGTCCTTCGACCAAA 313

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

Sbjct 335 TATGCCAGAGCTGAAGGACAGAACACCGGCAACTTCCTCACTGACCGTCCTTCGACCAAA 394

Query 314 GTTCACGCAGCTCCGGGAGGAGGATCATCCTTGGATTATCTCTTCACTGGTGGCAAGTAA 373

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

Sbjct 395 GTTCACGCAGCTCCGGGAGGAGGATCATCCTTGGATTATCTCTTCACTGGTGGCAAGTAA 454

>gb|AY464947.1| Arabidopsis thaliana spiral1 (SPR1) mRNA, complete cds

Length=646

GENE ID: 814896 SPR1 | spiral1 [Arabidopsis thaliana]

(10 or fewer PubMed links)

Score = 628 bits (340), Expect = 2e-178

Identities = 350/360 (97%), Gaps = 0/360 (0%)

Strand=Plus/Plus

Query 14 ATGGGTCGTGGAAACAGCTGTGGTGGAGGTCAAAGCTnnnnnnnnnnTCTCTTTGGTGGT 73

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

Sbjct 82 ATGGGTCGTGGAAACAGCTGTGGTGGAGGTCAAAGCTCATTGGATTATCTCTTTGGTGGT 141

Query 74 GACGCTCCTGCTCCTAAGCCAGTTCCAGCTCCTCGTCCCGCTCCTACTGAGTCTAACAAC 133

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

Sbjct 142 GACGCTCCTGCTCCTAAGCCAGTTCCAGCTCCTCGTCCCGCTCCTACTGAGTCTAACAAC 201

Query 134 GGACCTGCACCACCAGTAACAGCTGTGACTGCAACCGCACTCACGACTGCTACTACTTCT 193

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

Sbjct 202 GGACCTGCACCACCAGTAACAGCTGTGACTGCAACCGCACTCACGACTGCTACTACTTCT 261

Query 194 GTTGAGCCTGCAGAGCTTAACAAGCAGATTCCTGCTGGTATCAAAACTCCTGTTAACAAC 253

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

Sbjct 262 GTTGAGCCTGCAGAGCTTAACAAGCAGATTCCTGCTGGTATCAAAACTCCTGTTAACAAC 321

Query 254 TATGCCAGAGCTGAAGGACAGAACACCGGCAACTTCCTCACTGACCGTCCTTCGACCAAA 313

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

Sbjct 322 TATGCCAGAGCTGAAGGACAGAACACCGGCAACTTCCTCACTGACCGTCCTTCGACCAAA 381

Query 314 GTTCACGCAGCTCCGGGAGGAGGATCATCCTTGGATTATCTCTTCACTGGTGGCAAGTAA 373

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

Sbjct 382 GTTCACGCAGCTCCGGGAGGAGGATCATCCTTGGATTATCTCTTCACTGGTGGCAAGTAA 441