WSSP Chapter 9
Determine ORF and BLASTP
atttaccgtg
tgatgagtat
ccggaaatag
acttcaatga
ttggattgaa
gatacagttt
gatcccgatc
ttggttctaa
attatcttgc
tccgtattaa
atgattgctt
gcattcgaat
atgagccagc
taacgaacgg
caatattttc
gcgtacccgt
Steps and terms used in protein expression
1st ATG in mRNA
p 9-1
Cloning the cDNA library
p 9-1
Possible reading frames
p 9-2
Possible types of clones in the cDNA library
p 9-2
DSAP Define ORF page:
Link to Toolbox translation program
p 9-3
Toolbox: DNA Sequence Translation Program
PolyA tail at 3’ end
Reading frames
p 9-3
EX1.12 +1 Reading Frame
Longest ORF
Translation stop
p 9-3
Which one of these would be the correct ORF?
A)
B)
Rule #1: If downstream of a stop codon, translation
of the protein MUST start with an M (MET)
p 9-3
Could this ORF code for the protein?
?
p 9-4
Does this region match the BLASTX matches?
Region of DNA
that codes for
the highlighted in
protein sequence
BLASTx
p 9-4
Could the DNA code for a partial protein?
?
p 9-4
Does this region match the BLASTX matches?
Region of DNA
that codes for
the highlighted in
protein sequence
BLASTx
p 9-4
Does this region match the BLASTX matches?
Region of DNA
that codes for
the highlighted in
protein sequence
BLASTx
p 9-4
An example of a partial coding sequence
Similar
Seq.
Is this a partial ORF cDNA clone?
What about
this region?
The first part of the protein may not have
matches because it is not conserved.
Query
Sbjct
2
60
410
Region of similarity
475
The BLASTx helps determine which reading frame is correct
>ref|NP_001150519.1| dynein light chain LC6, flagellar outer arm [Zea mays]
Length=93
Score = 158 bits (400), Expect = 5e-37
Identities = 73/93 (78%), Positives = 83/93 (89%), Gaps = 0/93 (0%)
Frame = +2
Query
11
Sbjct
1
Query
191
Sbjct
61
MLEGRARVEDTDMPRKMQAEAMNAASHALDLFDVADCKSLAAHIKKEFDKIYGPGWQCVV
MLEG+A VEDTDMP KMQA+AM+AAS ALD FDV DC+S+A+HIKKEFD I+GPGWQCVV
MLEGKAVVEDTDMPAKMQAQAMSAASRALDRFDVLDCRSIASHIKKEFDAIHGPGWQCVV
GSSFGCFFTHKKGSFIYFRLETLHFLIFKGAAA
GS FGC+ TH KGSFIYFRLE+L FL+FKGAAA
GSGFGCYITHSKGSFIYFRLESLRFLVFKGAAA
190
60
289
93
It also helps suggest the start point
p 9-6
Chose the reading frame and paste in the protein sequence
Do not
include the *
(stop codon)
Make sure
to include
bases that
code for the
stop codon
p 9-7
The Five Commandments of DSAP
I. The stop codon is part of the ORF
DSAP BLASTp page
p 9-8
NCBI BLASTp page
Paste in protein sequence
p 9-8
BLASTp results of EX1.12 +2 ORF
Link to
Conserved
Domain
Database
p 9-9
BLASTp results of EX1.12 +1 ORF
BLASTp results of EX1.12 +3 ORF
No matches
Enter BLASTp data into table
Protein
Possible
DNA
Clones
M
*
AAAAAA
AAAAAA
AAAAAA
p 9-10
Suppose the cDNA was missing the first 13 bp
Does this DNA code for the start of the protein?
>gi|226493894|ref|NP_001150519.1| dynein light chain LC6, flagellar outer arm [Zea
mays]
Length=93 Score = 139 bits (351), Expect = 8e-32 Identities = 63/81 (77%),
Positives =
Query
1
Sbjct
13
Query
61
Sbjct
73
MPRKMQAEAMNAASHALDLFDVADCKSLAAHIKKEFDKIYGPGWQCVVGSSFGCFFTHKK
MP KMQA+AM+AAS ALD FDV DC+S+A+HIKKEFD I+GPGWQCVVGS FGC+ TH K
MPAKMQAQAMSAASRALDRFDVLDCRSIASHIKKEFDAIHGPGWQCVVGSGFGCYITHSK
GSFIYFRLETLHFLIFKGAAA
GSFIYFRLE+L FL+FKGAAA
GSFIYFRLESLRFLVFKGAAA
81
93
60
72
Suppose the cDNA was missing the first 13 bp
Did they choose the correct ORF?
>gi|226493894|ref|NP_001150519.1| dynein light chain LC6, flagellar outer arm [Zea
mays]
Length=93 Score = 139 bits (351), Expect = 8e-32 Identities = 63/81 (77%),
Positives =
Query
1
Sbjct
13
Query
61
Sbjct
73
MPRKMQAEAMNAASHALDLFDVADCKSLAAHIKKEFDKIYGPGWQCVVGSSFGCFFTHKK
MP KMQA+AM+AAS ALD FDV DC+S+A+HIKKEFD I+GPGWQCVVGS FGC+ TH K
MPAKMQAQAMSAASRALDRFDVLDCRSIASHIKKEFDAIHGPGWQCVVGSGFGCYITHSK
GSFIYFRLETLHFLIFKGAAA
GSFIYFRLE+L FL+FKGAAA
GSFIYFRLESLRFLVFKGAAA
81
93
60
72
Suppose the cDNA was missing the first 13 bp
Did they choose the correct ORF?
BLASTP starting here
>gi|226493894|ref|NP_001150519.1| dynein light chain LC6, flagellar outer arm [Zea mays]
Length=93 Score = 139 bits (351), Expect = 8e-32 Identities = 63/81 (77%), Positives =
Query
1
Sbjct
13
Query
61
Sbjct
73
MPRKMQAEAMNAASHALDLFDVADCKSLAAHIKKEFDKIYGPGWQCVVGSSFGCFFTHKK
MP KMQA+AM+AAS ALD FDV DC+S+A+HIKKEFD I+GPGWQCVVGS FGC+ TH K
MPAKMQAQAMSAASRALDRFDVLDCRSIASHIKKEFDAIHGPGWQCVVGSGFGCYITHSK
GSFIYFRLETLHFLIFKGAAA
GSFIYFRLE+L FL+FKGAAA
GSFIYFRLESLRFLVFKGAAA
81
93
BLASTP starting here
>gi|226493894|ref|NP_001150519.1| dynein light chain LC6, flagellar outer arm [Zea mays]
Score = 156 bits (395), Expect = 6e-37
Identities = 72/92 (78%), Positives = 82/92 (89%), Gaps = 0/92 (0%)
Query
1
Sbjct
2
Query
61
Sbjct
62
LEGRARVEDTDMPRKMQAEAMNAASHALDLFDVADCKSLAAHIKKEFDKIYGPGWQCVVG
LEG+A VEDTDMP KMQA+AM+AAS ALD FDV DC+S+A+HIKKEFD I+GPGWQCVVG
LEGKAVVEDTDMPAKMQAQAMSAASRALDRFDVLDCRSIASHIKKEFDAIHGPGWQCVVG
SSFGCFFTHKKGSFIYFRLETLHFLIFKGAAA
S FGC+ TH KGSFIYFRLE+L FL+FKGAAA
SGFGCYITHSKGSFIYFRLESLRFLVFKGAAA
92
93
60
61
60
72
Compare the BLASTx and BLASTp results for EX1.12:
Are the matches to the same proteins?
p 9-11
Compare the BLASTx and BLASTp results for
EX1.12: Are the e-values similar?
p 9-12
Compare the BLASTx and BLASTp results for
EX1.12: Are the alignments similar?
BLASTx
>ref|NP_001150519.1|
Length=93
Query
11
Sbjct
1
Query
191
Sbjct
61
dynein light chain LC6, flagellar outer arm [Zea mays]
MLEGRARVEDTDMPRKMQAEAMNAASHALDLFDVADCKSLAAHIKKEFDKIYGPGWQCVV
MLEG+A VEDTDMP KMQA+AM+AAS ALD FDV DC+S+A+HIKKEFD I+GPGWQCVV
MLEGKAVVEDTDMPAKMQAQAMSAASRALDRFDVLDCRSIASHIKKEFDAIHGPGWQCVV
GSSFGCFFTHKKGSFIYFRLETLHFLIFKGAAA
GS FGC+ TH KGSFIYFRLE+L FL+FKGAAA
GSGFGCYITHSKGSFIYFRLESLRFLVFKGAAA
190
60
289
93
BLASTp
>gi|226493894|ref|NP_001150519.1| dynein light chain LC6, flagellar outer arm [Zea mays]
Length=93 Score = 158 bits (400), Expect = 2e-37
Query
1
Sbjct
1
Query
61
Sbjct
61
MLEGRARVEDTDMPRKMQAEAMNAASHALDLFDVADCKSLAAHIKKEFDKIYGPGWQCVV
MLEG+A VEDTDMP KMQA+AM+AAS ALD FDV DC+S+A+HIKKEFD I+GPGWQCVV
MLEGKAVVEDTDMPAKMQAQAMSAASRALDRFDVLDCRSIASHIKKEFDAIHGPGWQCVV
GSSFGCFFTHKKGSFIYFRLETLHFLIFKGAAA
GS FGC+ TH KGSFIYFRLE+L FL+FKGAAA
GSGFGCYITHSKGSFIYFRLESLRFLVFKGAAA
60
60
93
93
p 9-12
DSAP Review Page
DSAP Review Page
p. 7-17
Use Toolbox to
determine the ORF
Do NOT use
Toolbox to
determine
the 5’
UTR!!!
The Five Commandments of DSAP
I. The stop codon is part of the ORF
II. The start of the 5’ UTR is always the
first base
Do NOT use
Toolbox to
determine
the 3’ UTR!!!
Determine ranges of 5’ UTR and 3’ UTR by
highlighting the ranges in the DSAP cDNA text box
p. 9-14
What should you do
if your clone is a
partial?
An example of a partial coding sequence
Similar
Seq.
The first bases are part of the reading frame
?
S I R
XGC TCA ATC CGT
The Five Commandments of DSAP
I. The stop codon is part of the ORF
II. The start of the 5’ UTR is always the
first base
III. If the clone is a partial, there is no 5’
UTR
IV. If the clone is a partial, the start of
the ORF is always the first base
What
should you
do if you
get these
results?
BLASTX
Why is my cDNA noncoding?
Genomic
DNA
RNA
cDNA
(Partial)
ORF
AAAAAAA
AAAAAAA
Recent genome wide RNA sequence studies show that
more than 10% of polyA RNAs are non-coding
If your DNA
is noncoding,
enter in the
entire
sequence
as 3’ UTR
The Five Commandments of DSAP
I. The stop codon is part of the ORF
II. The start of the 5’ UTR is always the
first base
III. If the clone is a partial, there is no 5’
UTR
IV. If the clone is a partial, the start of
the ORF is always the first base
V. If the clone is non-coding, the
entire DNA 3’ UTR
The Five Commandments of DSAP
I. The stop codon is part of the ORF
II. The start of the 5’ UTR is always the first base
III. If the clone is a partial, there is no 5’ UTR.
IV. If the clone is a partial, the start of the ORF is
always the first base.
V. If the clone is non-coding, the entire DNA 3’ UTR