WSSP-10 Chapter 6
1. Analysis of the REV waveform
2. Determine if the FOR and REV sequences overlap
3. Making a contig from the FOR-REV Sequences
Possible FOR and REV sequence overlaps
p. 6-1
If the For sequence does not span the insert request
that the clone be sequenced in the REV direction
p. 6-1
Download both sequences
p. 6-2
Crop the For sequence as in Chapter 4
Crop the 3’ end further out
p. 6-2
The REV sequence must go through a run of Ts.
p. 6-2
Sequences after long runs of Ts may be bad
p. 6-2
Slipping of strands during DNA synthesis
Length of DNA
5'-TTTTTTTTTTTTTC
N
3'-AAAAAAAAAAAAAGCCCTGCTCC-5'
N+1
N-1
T
5'-TTTTTTTTTTTTTC
3'-AAAAAAAAAAAAAGCCCTGCTCC-5'
5'-TTTTTTTTTTTTTC
3'-AAAAAAAAAAAAAGCCCTGCTCC-5'
A
N
N-1
N+1
Indicate in DSAP if the REV sequence is good or bad
p. 6-2
Map of the pTriplEX2 polylinker (MCS)
p. 6-3
Determining the start of the insert of a Rev sequence
Start of the insert
p. 6-4
To find the end of the insert, look for SmaI that is part
of the vector, then work your way back to the start
p. 6-5
Make sure it is the SmaI site in the vector and not the insert
??
p. 6-5
The EX2.10 REV sequence does not span to the vector
Enter positions into DSAP then crop ends
p. 6-5
II. Determine if the For and Rev sequences overlap
p. 6-6
Make the reverse complement of the REV sequence
p. 6-6
Normal orientation of an REV sequence
Reverse complement of an REV sequence
p. 6-7
Paste the REVrc sequence into the DSAP text box
p. 6-8
Determine if the sequences overlap
p. 6-8
NCBI BLAST Home page
p. 6-9
BLAST two sequences (alignment) tool
p. 6-9
Alignment of the EX2.10 FOR and REVrc sequences
p. 6-10
Sequence alignment between the FOR and REVrc sequences
p. 6-10
No alignment between two sequences
p. 6-11
Determine the start of the overlap
p. 6-11
3’ ends of FOR and REV sequences may be unreliable
How bad are they?
Examine and fix
mismatches
Edit difference between the two sequences
p. 6-12
Determine
which
sequence is
more likely to
be correct at
each mismatch
p. 6-14
Make a contig (composite between the For and Rev sequences
Use one sequence if there is a complete overlap
p. 6-13
Crop overlapping regions
p. 6-13
EX2.10 sequences after cropping at the junction
p. 6-13
Check your work for gaps
Indication the contig was not
made properly (extra C)
p. 6-14
Enter contig sequence into DSAP and perform the rest of
the analysis as was done for a For sequence
p. 6-14