Biomedical Engineering Reference
In-Depth Information
7 Under 'Sequence Formatting Options' choose 'Exons in upper case, everything else in
lower case.'
8 Click the 'Mask Repeats' checkbox and set the radio button, 'to N.'
g
9 Click the 'Submit' button.
10 In a separate internet browser window, navigate to the Primer3Plus web tool.
11 Under Task, select 'Sequencing' from the pull-down menu.
12 Paste the target gene sequence into the sequence field. Using brackets ([ ]), mark the
region or regions of interest within the gene sequence.
h
13 Click the 'General Settings' tab and set the following options: Primer Size - Min
=
20
Opt
=
23 Max
=
26; Primer TM - Min
=
54 Opt
=
55 Max
=
56; Primer GC% - Min
=
20.0 Max
=
50.0. Select the appropriate Mispriming/Repeat Library.
i
14 Adjust the Advanced Settings. Set Product Size Min
=
200, Opt
=
400, Max
=
600.
Under the 'Sequencing' section of the Advanced Settings tab, set: Spacing
=
500,
Interval
=
400, Lead
=
50, Pick Reverse Primers
=
Checked.
15 Click the Pick Primers button.
j
16 If primer design succeeds, the primer selections will be highlighted in your sequence.
Scroll down to see the primer sequences. Check the box next to the desired primers to
and click the 'Send to Primer3Manager' button.
17 Check the primers with the Primer3Manager. For each primer, verify that TM is between
54 and 56
◦
C and that GC content is between 20 and 50%.
Notes
a
Available from http://genome.ucsc.edu/ [10].
b
Available from http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi [15].
c
If the sequence for PCR primer design is already available then proceed to step 10.
d
A list of genes should appear in table form. Click on the required gene description to bring up
the summary page.
e
This should bring the 'Get Genomic Sequence Near Gene' page.
f
Generally, these should include 5' UTR exons, CDS exons and 3' UTR exons.
g
This will prevent the design of primers in repetitive regions, and also make the target (exon)
sequence easy to identify.
h
This can also be performed by highlighting the desired sequence with the mouse and then
clicking the brackets button ([ ]) next to 'Mark selected region.'
i
Repeat/Mispriming libraries are species specific. Select the library for the species of interest.
j
The next page may take a few minutes to load.
2.2.2.4 Targeted resequencing of genomic DNA
Once samples have been chosen, targets identified, and requisite PCR primers designed, it
is time to proceed to the actual DNA sequencing. As the sequencing reads will be utilized
for variant discovery, obtaining the highest quality data is crucial. Ideally, PCR amplicons
