Download p. 6-23

WSSP Chapter 7
BLASTN: DNA vs DNA searches
atttaccgtg
tgatgagtat
ccggaaatag
acttcaatga
ttgattaata
ttggattgaa
gatacagttt
gatcccgatc
ttggttctaa
tttccatttc
attatcttgc
tccgtattaa
atgattgctt
gcattcgaat
tgtcccagtt
atgagccagc
taacgaacgg
caatattttc
gcgtacccgt
tttaattttc
4-3
DSAP: BLASTn Page
p. 7-1
NCBI BLAST Home Page
p. 7-1
NCBI BLASTN search page
p. 7-2
Copy sequence from DSAP or wave form program
p. 7-2
Choose a database (nr/nt or est)
p. 7-3
Search options (Use defaults)
p. 7-4
BLASTN progress report (search may take a few minutes)
p. 7-5
Format options (use defaults)
p. 7-5
EX1.11 BLASTN nr/nt database
p. 7-6
Graphic report of EX2.09
p. 7-7
BLASTN list of matches for EX1.10
p. 7-7
EX2.09
BLASTN
p. 7-9
Best match to EX1.12
Length of
sequence
Our Seq.
Database
Seq.
Mismatch
Match
>gi|226493893|ref|NM_001157047.1|
outer arm (LOC100284150), mRNA
Length=606
Zea mays dynein light chain LC6, flagellar
Score = 221 bits (244), Expect = 5e-54
Identities = 218/282 (77%), Gaps = 0/282 (0%)
Strand=Plus/Plus
Query
11
Sbjct
104
Query
71
Sbjct
164
Query
131
Sbjct
224
Query
191
Sbjct
284
Query
251
Sbjct
344
ATGTTGGAAGGGAGGGCGAGAGTAGAAGACACCGACATGCCGAGGAAGATGCAGGCGGAG
||||||||||| | ||||
|| || ||||||||||||||| ||||||||| || ||
ATGTTGGAAGGAAAGGCGGTGGTGGAGGACACCGACATGCCGGCGAAGATGCAAGCCCAG
70
GCCATGAACGCCGCCTCTCACGCGCTCGATCTGTTCGACGTCGCGGACTGCAAGAGCCTC
|| |||
|| || ||
|| || |||| |||||||||
|||||| |||| ||
GCGATGTCGGCGGCGTCCAGGGCCCTGGATCGCTTCGACGTCCTCGACTGCCGGAGCATC
130
GCCGCGCATATCAAGAAGGAATTTGATAAGATCTACGGTCCGGGATGGCAGTGCGTCGTC
|| | || ||||||||||| |||||
|||| | || || |||||||| ||||| ||
GCGTCCCACATCAAGAAGGAGTTTGACGCGATCCATGGCCCCGGATGGCAATGCGTGGTT
190
GGCTCCAGCTTCGGCTGTTTCTTCACTCACAAGAAAGGCAGCTTCATCTACTTCCGCCTG
|||||| |||||||||| | | |||| |||| || || |||||||||||||||||||||
GGCTCCGGCTTCGGCTGCTACATCACGCACAGCAAGGGGAGCTTCATCTACTTCCGCCTG
250
GAGACGCTCCACTTCCTCATCTTCAAAGGCGCGGCCGCTTGA
||| |||||
|||||| |||||||||| ||||| || |||
GAGTCGCTCAGGTTCCTCGTCTTCAAAGGGGCGGCAGCATGA
163
223
283
343
292
385
p. 7-9
Perfect, but short, matches are not
usually meaningful
>gi|14250883|emb|AL583809.3|CNS07EFY Human chromosome 14 DNA
sequence BAC R-736L22 of library RPCI-11 from chromosome 14 of
Homo sapiens (Human), complete sequence
Score = 40.1 bits (20), Expect = 4.6 Identities = 20/20 (100%)
Query: 189
ttttctgaatattcataata 208
||||||||||||||||||||
Sbjct: 60645 ttttctgaatattcataata 60626
7-11
Examine the best alignments:
Are they significant?
7-9
Mismatches
i)
Bad sequence on our part
ii)
Bad sequence on their part
iii) Differences in the sequence of the two organisms
Query
Sbjct
C
TGT
|||
TGT
C
R
CGT
|||
CGT
R
E
GAA
|||
GAA
E
L
CTC
||
CTT
L
L
CTA
||
CTG
L
I
ATT
||
ATC
I
L
CTC
||
CTT
L
D
GAC
||
GAT
D
A
GCC
||
GCA
A
Wobble position: same amino acid,
but different codon….degenerate code
Query:
Sbjct:
383 AGCGTTGCCGTTCGTCAGCTTGATGTTAAGCTGGGCAGCGCGCTCGACGATTCCTTTGCG 324
|||||| |||||||||||||||||||| | ||| || ||||||||||||||||| |||||
6152 AGCGTTTCCGTTCGTCAGCTTGATGTTCAACTGAGCGGCGCGCTCGACGATTCCCTTGCG 6211
Small Gaps- alter the reading frame of the protein
Query
Sbjct
C R
R T P D P *
TGTCGT-CGAACTCCTGATCCTTGA
|||||| ||||||||||||||||||
TGTCGTCCGAACTCCTGATCCTTGA
C R E L L I L D
p. 7-13
An example of a match with and without gaps.
Query:
Sbjct:
Query:
Sbjct:
179 TTCGAGCTACCAGATGATC-GATTGGAACAT-T-C--TGTCATTG-AC-CTTC-AGGTAA 230
||||||| || | | || |||| || || | | | | ||| | |||| |||| |
4684 TTCGAGCG-CC-GTTAATATGATTACAATATCTACAATATTATTATATGCTTCCAGGTGA 4741
231 TCAACCATGACCGTGTCAACCGAAACGACGTTATCGGCCGTGCACTATTGAACATGGAGG 290
|||| ||||||||||| ||||| || || || || |||||||| || | || ||||| |
4742 TCAATCATGACCGTGTTAACCGTAATGATGTAATTGGCCGTGCCCTTCTTAATATGGAAG 4801
p. 7-13
Alignment of the second best match to EX1.12
>gi|241990611|dbj|AK330768.1| Triticum aestivum cDNA, clone: SET5_E05, cultivar:
Chinese Spring Length=650
Score = 219 bits (242), Expect = 2e-53
Identities = 211/271 (77%), Gaps = 0/271 (0%)
Query
10
Sbjct
78
Query
70
Sbjct
138
Query
130
Sbjct
198
Query
190
Sbjct
258
Query
250
Sbjct
318
GATGTTGGAAGGGAGGGCGAGAGTAGAAGACACCGACATGCCGAGGAAGATGCAGGCGGA
|||| ||||||||| ||||| || || |||||||||||||||
||||||||| | |
GATGCTGGAAGGGAAGGCGACGGTGGAGGACACCGACATGCCGGCCAAGATGCAGCTGCA
69
GGCCATGAACGCCGCCTCTCACGCGCTCGATCTGTTCGACGTCGCGGACTGCAAGAGCCT
|||||
|| || ||
|||||||| | |||||||||
|||||| |||| |
GGCCACCTCGGCGGCGTCCAGGGCGCTCGAACGCTTCGACGTCCTCGACTGCCGGAGCAT
129
CGCCGCGCATATCAAGAAGGAATTTGATAAGATCTACGGTCCGGGATGGCAGTGCGTCGT
||| ||||| ||||||||||| || || | |||| |||| ||||| ||||||||||| ||
CGCGGCGCACATCAAGAAGGAGTTCGACACGATCCACGGCCCGGGGTGGCAGTGCGTGGT
189
CGGCTCCAGCTTCGGCTGTTTCTTCACTCACAAGAAAGGCAGCTTCATCTACTTCCGCCT
|||| |||||||||||| | |||||| |||| || || |||||||| ||||||
||
GGGCTGCAGCTTCGGCTGCTACTTCACGCACAGCAAGGGGAGCTTCATATACTTCAAGCT
249
GGAGACGCTCCACTTCCTCATCTTCAAAGGC
||| |||||| |||||| |||||||||||
CGAGTCGCTCCGGTTCCTCGTCTTCAAAGGC
137
197
257
317
280
348
p. 7-14
Alignments near the end of the EX1.12
>gi|254826767|ref|NG_012498.1| Homo sapiens glypican 4 (GPC4),
RefSeqGene on chromosome X Length=121142 Score = 71.6 bits (78),
Expect = 6e-09 Identities = 42/44 (95%), Gaps = 0/44 (0%)
Query
665
Sbjct
72886
CTAGCTTTTCTTAACaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
|| ||||||||||| |||||||||||||||||||||||||||||
CTTGCTTTTCTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
708
72929
p. 7-14
Fill in the table listing the best matches from
three different organisms.
List Wolffia if there is a match
p. 7-15
Use the
clone report
to obtain
more
information
about the
gene
p. 7-15
3) Perform a
BLASTn of
the est
database
Change
the
database
p. 7-17
BLASTn report of the EX1.11 search
of the est database
p. 7-17
Alignment of the best match to EX1.12
from the est search
>gi|198335694|gb|GD004539.1| CCHY28888.g1 CCHY Panicum virgatum callus (N) Panicum virgatum
cDNA clone CCHY28888 3', mRNA sequence. Length=624
Score = 246 bits (272), Expect = 1e-61
Identities = 226/286 (79%), Gaps = 0/286 (0%)
Strand=Plus/Minus
Query
3
Sbjct
527
Query
63
Sbjct
467
Query
123
Sbjct
407
Query
183
Sbjct
347
Query
243
Sbjct
287
GAGAGAAGATGTTGGAAGGGAGGGCGAGAGTAGAAGACACCGACATGCCGAGGAAGATGC
|||| | ||| ||||||||| ||||| || || ||||| ||||||||| ||||||||
GAGACACCATGCTGGAAGGGAAGGCGATGGTGGAGGACACGGACATGCCGGCGAAGATGC
62
AGGCGGAGGCCATGAACGCCGCCTCTCACGCGCTCGATCTGTTCGACGTCGCGGACTGCA
||||| |||| |||
|| || ||
|| ||||| | |||||||||
||||||
AGGCGCAGGCGATGGCGGCGGCGTCCAGGGCCCTCGACCGCTTCGACGTCCTCGACTGCC
122
AGAGCCTCGCCGCGCATATCAAGAAGGAATTTGATAAGATCTACGGTCCGGGATGGCAGT
|||| |||| ||||| ||||||||||| ||||| | |||| |||| || || ||||| |
GGAGCATCGCGGCGCACATCAAGAAGGAGTTTGACACGATCCACGGCCCCGGGTGGCAAT
182
GCGTCGTCGGCTCCAGCTTCGGCTGTTTCTTCACTCACAAGAAAGGCAGCTTCATCTACT
|||| || ||||||||||||||||| | |||||| |||| || || |||||||||||||
GCGTGGTGGGCTCCAGCTTCGGCTGCTACTTCACGCACAGCAAGGGGAGCTTCATCTACT
242
TCCGCCTGGAGACGCTCCACTTCCTCATCTTCAAAGGCGCGGCCGC
|||| || ||| |||||
||||||||||||||||| ||||| ||
TCCGGCTCGAGTCGCTCAGGTTCCTCATCTTCAAAGGGGCGGCAGC
468
408
348
288
288
242
p. 7-17
Fill out the DSAP table of the BLASTn
search of the est database
p. 7-18
Open Question: Why are there differences in the sequences?
Query
61
Sbjct
13166
Query
121
Sbjct
13106
CAAGGTCTAAGTACTGAAAAGGAAAGTCTACTAATTACAAAGAAGTTATTGTTTGTACCT
|||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||
CAAGGTCTAAGTACTGAAAAGGAAAGTCCACTAATTACAAAGAAGTTATTGTTTGTACCT
120
TTTGTATCAGGGTTTATTAAATTTCAATCTTTATTGCTGAATCCCGAAACAAGGTGATCT
|||||||||||||||||||||||| |||||| ||||||||||||||||||||||||||||
TTTGTATCAGGGTTTATTAAATTTTAATCTTCATTGCTGAATCCCGAAACAAGGTGATCT
180
13107
13047
Q5. BLASTn Analysis: Is your cDNA similar to genes in
other organisms?
p. 6-23
Q6. BLASTn Analysis: Is your cDNA similar to genes in
many different organisms?
p. 6-23
Is the sequence found in many other organisms?
!