Our
APA DNA walking service is a sensitive, specific and fast PCR-based method that
will help in fulfilling your goal:
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Gap
filling.
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cDNA
walking.
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5'
promoter region and 3' region walking.
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Identify
viral insertion/genomic, intron/exon, or transgene/genomic DNA junctions.
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Amplify
upstream and downstream adjacent sequences based on known sequences.
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Bi-directional
sequence extension of STSs and EST.
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Identify
gene traps and transposon-insertion locations.
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End-sequencing
or walking of BAC/PAC DNA.
Based
on known sequences, we can amplify upstream and downstream sequences by
multiple APA walking. Our experimental design to achieve this result is
delineated below:
Prior
to walking step
Confirmation
of known sequences by Bio S&T:
-
Client
has to provide
a pair of primers
and a
DNA template
in order to amplify a known sequence (preferably 150-1000bp). Bio S&T will
perform the PCR and sequence the obtained amplicon before proceeding with the
walking. If the obtained sequence is as expected, Bio S&T will carry out
the walking service. Client must provide expected amplicon sequence for
confirmation. If the obtained sequence differs from the expected sequence, Bio
S&T will abort project. This step entails a
non-refundable $150.00 fee.
Or
-
Should
the client not have a primer pair to provide, Bio S&T design 1-2 pairs of
primers based on the client-provided known sequence. Bio S&T will amplify a
150-300 bp fragment and sequence it.
This step entails a non-refundable $300.00 fee.
Walking
step
-
Design and synthesis of primers:
normally 3-4 primers are designed according to the known starting sequences.
The fidelity of the known sequence is the most important ingredient for the
success of any kind of PCR-based genomic walking method, including APA.
-
PCR amplification and sequencing:
APAgene technology will be employed to amplify the upstream and/or downstream
sequences. The PCR products will be purified and/or cloned, followed by
sequencing. The customer will receive the newly acquired flanking sequences
from a successful walk. The overlapped sequences will be at least 20bp in
length between each walk. The primers will be redesigned, and PCR carried to
get any length of sequence required. In
some cases, the sequences generated by APA walking are not in accordance with
client’s expectations. Should there be disaccord, Bio S&T will design an
outer primer based on the newly obtained sequences. PCRs will be carried out
with this primer and one gene-specific walking primer in order to amplify an
overlapped fragment. If the obtained size is correct, the walking will be
deemed successful. An additional $150.00 will be charged for this step.
However, if the test fails to amplify the correct overlap fragment, the
walking result will be deemed as false positive and client will not be charged
for the walking step or this test step.
-
Cost:
$500.00 per walk (per direction) for the
first 250bp plus
$1.00/base for the newly acquired
sequences beyond the initial 250 bp. Walking will be attempted
twice. Should walking fail (no sequence information is obtained), client will
be invoiced $200.00 (per direction) as a
setup-fee (in addition to confirmation fee). This
fee is non-refundable.
-
Sample requirements:
We accept genomic DNA, cDNA or plasmid DNA (such as BAC/PAC). 20
microliters suitable for Southern blot and PCR, intact and devoid of any
contamination, should be provided. Amount required per kilo base
walked: source genomic DNA = 50ng/ul, source cDNA = 10-20ng/ul, source
plasmid DNA = 2-5ng/ul. We are aware that some DNA samples from customers are
mixed with other DNA sources, such as mixed DNA samples resulting from F1
segregation of DNA. In case a contaminated sample is sent, every successful run
as defined before will be charged, although the sequence generated may be
contrary to expected result. DNA purification from tissues may also be
performed at our facility for $200.00.
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Starting sequences:
We need 150 bp or more to design suitable primers. For starting sequences
of less than 150bp, a non-refundable $100.00 set-up fee will apply (this
setup-fee is not applicable under other circumstances). We require confirmation
of the starting sequences. Please note that an incorrect starting sequence
(even a one base error!), can cause the failure of a PCR-based walking
method.
-
Deliverables:
Sequence data will be provided via email.
-
Please note:
We do not accept project with multiple tandem repeats as the internal repeats
are always preferentially amplified (i.e. failure rate is high).
Should any technical difficulties arise, Bio S&T will provide written or
verbal notice to the client. Bio S&T also reserves the right to re-quote
this project if the scope of the project changes, in which event Bio S&T
will not be required to perform the project unless both parties can agree upon
the terms of the revised quotation.
Please
contact us fro more information or to place an order!
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