- [Instructor] This video will show you
how to design LAMP primers
using the New England Biolabs LAMP Primer Design Tool.
To get started
identify the DNA sequence
you want to design primers for
and select a region
of fewer than 2,000 bases
to input into the tool.
This can be loaded as a.txt file
or pasted directly into the tool
using the "Paste Sequence" feature.
As an example
I'm pasting in a region
of <> gene sequence
Once the sequence is pasted in
click the "Process Text" button
to properly format the sequence text.
If desired, give the sequence target a name
in the "Target Name" box
but this is not required.
The modify preferences box
allows for modification
of the design parameters.
Lengths, Tm, GC content
and many other factors can be adjusted
but for any new sequence
we strongly recommend
using the default settings
for the first attempt
and that's what we'll do here.
After the sequence is pasted and processed
click the "Continue" box
at the bottom of the first screen.
The next screen displays the target sequence
with the ability to specify fixed locations
for targeting with one of the LAMP primer regions.
This is not common
but if you need a specific sequence
to be contained between different primers
for a probe or beacon
or a site of variation
to be included outside a primer
you can do that here.
In most cases
we just click "Generate Primers"
at the bottom
to start the algorithm
identifying suitable LAMP primers.
Candidate LAMP primer sets
are immediately displayed
on the next screen
with their positions along
the target input highlighted
in the box at the top.
If you can't see the primers
just scroll to the right
as they may be spaced
far apart on the sequence.
Each primer set is laid out
in more detail below
with each set of four Core LAMP primers highlighted
in tables, identified by the corresponding ID number.
eight sequences are displayed
but only four of those
are the primers F3, B3, FIP and BIP.
This is because the long inner primers FIP and BIP
are composed of two distinct target regions
F2 and F1c for FIP
B2 and B1c for BIP.
You can see these regions each
on the target sequence layout
but they are spaced some distance apart
and the c sections are included
in the FIP and BIP primers
and inverted complements.
A big reason why
using the LAMP primer design tool is helpful.
Select one of the designed candidate sets
to move forward to Loop primer design.
Ranking of sets is by default deltaG
of dimer formation.
It's very easy to try different sets
and data won't be lost
so just pick one to move forward.
These generated Loop Primers
are not in the "Core" set
of four length primers
from the previous screen.
And are not required for LAMP
but do provide a significant boost to speed
and assay performance
and we strongly recommend
using them where possible.
Click "Generate Loop Primers".
It is possible that no Loop primers can be designed
for the candidate Core primer set
in which case a "No Primer Sets Available" box
will pop up.
If that happens
select the next bubble down
and try again.
The next screen shows candidate primer sequences identified
for the Loop F and Loop B primers.
It is possible only one of the two
will be identified.
If only one Loop can be designed
that is recommended over none
but it's worth trying to find a set with both
and in most cases that is possible.
In the case that only one primer is identified
we recommend going back to the "Core_Primers" tab
or to select another primer set
and selecting the next set of Core candidates
and trying Loop design again.
Loop primer candidates
are displayed in a list at the top
and you'll notice there's a lot of similarity
between the options.
Where up to 10 potential pairs are shown
many with identical Loop F sequences
and slightly different Loop Bs.
Choose the top option
or one further down the list
if you'd like increased GC
or another factor.
Select the bubble for the Loop set you like
and then click" Get Selected Primers".
The final screen will display
the full designed primer set
adding in the two Loop primers
to the previous four Core primers on the table.
All relevant information
and oligo sequences are on the table
with the Orange sequences representing
the oligonucleotides to order
from your desired provider.
Information can be copy/pasted
or you can "Download Primer Set"
and you'll be provided
with a tab-delimited .txt file directly compatible
with batch ordering through the IDT portal.
For any new LAMP assay
it is advisable to screen
more than one candidate primer set
to find the best performance
in terms of speed, sensitivity
and non-template amplification.
From the Results tab
you can either click "Select More Primer Sets"
or the "Core_Primers" tab
to go back to the candidate set page.
And repeat the selection
and Loop primer design steps.
Each Set can be downloaded individually
for easy ordering
or you can paste all of the designed primers
into another sheet or form.
Just take care
to keep each set together
as the names are generic primer IDs
and not associated with the set.
If you have questions
about using the LAMP primer design tool
or need any other technical support
please visit info@neb.com.