Bioinformatics/Site-directed mutagenesis exercise - web-3

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1 Οκτ 2013 (πριν από 4 χρόνια και 1 μήνα)

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Bio
informatics Exercises: Surfing the Web for DNA Sequences
(modified
1
/11/1
2
)


I’m almost completely self
-
taught on this matter, so you’ll be learning what I’ve learned. I still
have much to learn but hopefully some of this will help you get started in DN
A sequence
analysis.

It will complement

what you’ve learned in analyzing the sequences that you got from
your clones.


I’m going to illustrate a search that I did, and we’ll look at the results; you’ll then do a similar
search and obtain results.


A good w
eb site: Here’s the search that I did on the cyclin gene of the fruit fly,
Drosophila
melanogaster.

Go to:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi


Click on “Protein”



enter “Drosophila

cyclin” You end up with
1440
hits!



Here’s where it gets a bit complicated. We find the document, or subdocument, that has a
Drosophila

cyclin protein sequence. This then needs to be analyzed by the BLAST program for
sequence similarity to other known

sequences. I’ll find the
Drosophila
cyclin sequence, then
convert to the FASTA format. This then needs to be copied into the BLAST enter space.


Note that there are, conveniently located Blinks
-

these show searches that have already been
done. We’ll p
retend that this is a new sequence that we have found.


I’m using
BAA01628


This is a cyclin A gene, and is 491 amino acids in length.



I then copy this AA sequence, and

then go to BLAST, then go to protein
-
protein BLAST
(BLASTP)


I paste the results of the protein sequence into the blast space.


I then did a query, using BLAST
-

results to be shown.


We get 100 hits (because we asked for the best 100).


Your assignment:


Pick a protein that you’re interested in. Do a protein
-
protein alignment (BLAS
T), and print out
your results
-

no, not the whole thing
-




Try to pick out any interesting information about your protein, in terms of how similar it is to a)
other proteins in

the same organism
AND

b) the same protein in a much different, unrelated,
organism.




Site
-
directed mutagenesis (SDM)

Paper
Exercise


In this process, you are altering the DNA sequence of the gene, usually to determine the effect
upon function
.

I us
ed SDM to inactivate the
trpA

gene, in producing the mutants that I use for
my evolutionary potential studies. The
trpA

and
trpB

genes are on a plasmid, pWS1, which we’ll
discuss later.




Step 1:
In two separate reactions,
ampl
ify by
PCR the two 'halves' of the area to be mutated.

PCR I
:


FtrpA
>


5’NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3’
3’NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN5’



<
R
175

PCR II F
175>

5’NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3’


3’NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN5’





<
RtrpA

The F175 & R175 primers are complementary to the DNA at that region,
except for 2
-
3 base
changes that alter the amino acid at position (position
175

in this case)



Gel Purify PCR products I & II;
m
ix, amplify again w/o primers, 8 c
ycles; I and II initially act as
template and primers.


5’NNNNNNNNNNNNNNNXNNN
3’
------------------




-----------------------

NNXNNNNNNNNNNNNNNNN5


Spike the reaction with FtrpA & RtrpA and continue for an additional 25 cycles to amplify the
fragments

from III




Fig. 1

Plasmid pWS1

With
trpA

E49V: pRS201
-
2

With
trpA
D60N: pRS213

With
trpA
E49V, D60N: pRS202
-
5


FtrpA


5’NNNNNN
H
NNNNNNNN
X
NNNNNNNNNNN
S
NNNNN3’
3’NNNNNN
H
NNNNNNNN
X
NNNNNNNNNNN
S
NNNNN5



RtrpA

X= changes nucleotide; H= Hpa1 site, S= Sal1 site.


We then digest with HpaI and SalI, producing:


5’
H
NNNNNNNN
X
NNNNNNNNNNN
S
3’

3’
H
NNNNNNNN
X
NNNNNNNNNNN
S
5’


This fragment can then be ligated to the large fragme
nt of pWS1.


Your assignment:


Prepare primers for changing the glutam
ic acid

at position 49 to
a valine.


In doing these things, annotate the sequence shown below, by
indicating in
bold

the regions requested.


1) Identify the HpaI and SalI sites that fl
ank this gene.

2) Identify the start and stop codon
s

for trpA

3) Identify the codons for the site you wish to change

4) Prepare a forward and reverse primer for mutating your site,
again with a T
m

that is at least 65
o
C. Indicate your primers by repeating

them below the
sequence. The forward primers would be in the same sense; the for the reverse primers show the
sequence in 3’
-
5’ direction. I’ll illustrate below.

5)Prepare a forward and reverse primer for all of trp A. Make
sure that the T
m

is
no more t
han

65
o
C,

no less than 55
o
C

and that they produce fragments
that include the HpaI and SalI sites. See instructions for #4.


Some information:

Sal I:
gtcgac

HpaI:
gttaac


Sites for calculating Primer Tm’s
www.operon.com



Site for doing simple manipulation of your sequences (getting reverse complement, etc.):


http://bioinformatics.org/JaMBW/



Sequence extractor
http://www.bioinformatics.org/seqext/

puts
amino acid sequences with your sequence, and finds restriction
enzyme sites, etc.


Key sequence information around the
trpA

fragment in pWS1


This information is from the trpCBA gene, sequence, locus
U23499





gene

1385..2578


/gene="trpB"


CDS

1385..2578


/gene="trpB"


/codon_start=1


/transl_table=
11


/product="tryptophan synthase beta subunit"


/protein_id="
AAB60055.1
"


/db_xref="GI:775192"


/translation="MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQKDPEFQAQ


FNDLLKNYAGRPTALTKCQNITAGTNTTLYLKREDLLHGGAHKTNQVLGQALLAKRMG


KTEIIAETGAGQHG
VASALASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVH


SGSATLKDACNEALRDWSGSYETAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILE


REGRLPDAVIACVGGGSNAIGMFADFINETDVGLIGVEPGGHGIETGEHGAPLKHGRV


GIYFGMKAPMMQTEDGQIEESYSISAGLDF
PSVGPQHAYLNSTGRADYVSITDDEALE


AFKTLCLHEGIIPALESSHALAHALKMMRENPEKEQLLVVNLSGRGDKDIFTVHDILK


ARGEI"


gene


2578..3384


/gene="trpA"


CDS

2578..3384


/gene="trpA"


/codon_start=1



/transl_table=
11


/product="tryptophan synthase alpha subunit"


/protein_id="
AAB60056.1
"


/db_xref="GI:775193"


/translation="MERYESLFTQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAG


ADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEVLALIRQKHPTIPIGLLMY


ANLVFNKGIDEFYAECEKVGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADD


DLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPD

QVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKAFVQPMKAATRS"


The sequence numbers begin with a reference

sequence of the
trp

operon.





Example of showing primers for amplifying the 2341
-
2541 region, and a DraI
site: (HINT: sequence extractor!)


DraI


2341
t
ggacgcgct gattacgtgt ctattaccga cga
tgaa
gcc ctggaagcc
t ttaaa
acgct


5’
t
ggacgcgct gattacgtgt

3’


2401 ttgcctgcat gaagggatca tcccggcgct ggaatcctcc cacgccttgg cccatgcgct



2461 gaaaatgatg cgc
gaaaatc cggaaaaaga gcag
ctactg gtg
gttaac
c tttccggtcg










2521 cggcgataaa gacatcttca ccgttcacga tattttgaaa gcacgagggg aaatctg
atg


3’g
ccgctattt

ctgtagaagt

g

5’




2581 gaacgctacg aatctctgtt tacccagttg aaggagcgca aagaaggcgc attcgttcct



2641 ttcgtcaccc tcggtgatcc gggcattgag cagtcgttga aa
attatcga tacgctaatt




2701
gaagccggtg ct
gacgcgct ggagttaggc atc
cccttct ccgac
ccact ggcggatggc





2761
ccgacg
attc
aaaacgccac actgcgtgct tttgcggcgg gagtaacccc ggcgcagtgc



2821 tttgaggtgc tggcactcat tcgccagaag cacccgacca ttcccatcgg ccttt
tgatg



2881 tatgccaacc tggtgtttaa caaaggcatt gatgagtttt atgccgagtg cgagaaagtc



2941 ggcgtcgatt cggtgctggt tgccgatgtg cccgtggaag agtccgcgcc cttccgccag



3001 gccgcgttgc gtcataatgt cgcacctatc tttatttgcc cgcctaatgc cgacgatgat




3061 tt
gctgcgcc agatagcctc ttacggtcgt
ggttacacct atttgctgtc g
cgagcgggc




3121 gtgaccggcg cagaaaaccg cgccgcgtta
cccct
caatc

atctggttgc gaagc
tgaaa




3181 gagt
acaacg ctgcgcctcc attgcaggga tttggtattt ccgccccgga tcaggtaaaa




3241 gcagcgattg atgcaggagc
tgcgggc
gcg atttctggtt cggccatc
gt

taaaatcatc



3301 gagcaacata ttaatgagcc agagaaaatg ctggcggcac tgaaagcttt tgtacaaccg





3361 atgaaagcgg cgacgcgc
ag t
taattccac agccgccagt tccgctggcg gcattttaac



3421 tttctttaac gaagccggaa aaatcctaaa ttcatttatt atttatcttt ttaccgtttc



3481 gcttacccct gtcgatcgtc aacttacgtc ttttttccgc ccaacagtaa tataatcat
a



3541 caaattaatc ccgcaacata acaccagtaa aatcaataat tttctctaag tcacttattc



3601 ctcaggcaat tgttaataca tccagaacgt tcctcaaaat atatttttcc actttcttct













3661 cgttacgctt aatttgacta attctcatta acgactaatt ttaatgagt
g tcgac end of
trp
A fragment



pACYC continues (
215
2 in the pACYC 184 sequence)


2152 cgatgccct


2161 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg


2221 cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg



2281 tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg


2341 tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt


2401 tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc tacgtcttgc


2461 tggcgttcgc gacgcgag
gc tggatggcct tccccattat gattcttctc gcttccggcg


2521 gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac gaccatcagg


2581 gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcact ggaccgctga


2641 tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgg
gttggca tggattgtag


2701 gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca