APA walking

We have improved our technology and methods to better deal with multiple-copy integration of transgenes! Our APA walking service is a sensitive, specific and fast PCR-based method applicable towards many objectives:

  • Gap filling
  • cDNA walking
  • 5' promoter region and 3' region walking
  • Identify viral insertion/genomic, intron/exon, or transgene/genomic DNA junctions
  • Amplify upstream and downstream adjacent sequences based on known sequences
  • Bi-directional sequence extension of STSs and EST
  • Identify gene traps and transposon-insertion locations
  • 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 outlined below:

Step #1 - $99


Confirmation of known sequences by Bio S&T: Client has to provide a pair of primers andDNA 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 is non-refundable.

Step #2 - $169


Synthesis of walking primers: 1 primer set will be designed and synthesized for 5’-direction and/or 3’direction walking. A total of $169/direction will be charged for this step. This step is non-refundable.

Step #3 - $999 per library (per direction)


Construction of a walking library. Should you need both the 5’ and 3’ flanking sequences, the second walking library will be offered at $599. This step is non refundable.

Step #4 -$899/sample (<10 samples) or $699/sample (10 or more samples)


PCR walking using APATM Technology : Cost will be $899 for sequencing data of one successful walking. A walk is considered successful when at least 150bp of flanking sequence is obtained. The length of each flanking sequence will vary from 150bp to 500bp. Each successful walking will be charged.

Step #5- $299/sequence


We will design one primer based on the obtained sequence. In combination with one walking primer, we will use this primer pair to amplify a fragment of expected size. If unsuccessful, the obtained sequences will be regarded as false, and no charge for Step#4 will be applied. If promoter is present in multiple locations, each successful confirmation will be charged (upon approval)

Why will we succeed?

Our service is extremely reliable and very cost-efficient!

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. We always perform a confirmation step to ensure 100% accuracy prior to walking - an incorrect starting sequence (even a one base error!), can cause the failure of a PCR-based walking method. Our new APA PCR-based method has fewer limitations than the existing PCR-based method which translates into RELIABLE results.

Other walking methods rely on one or both of the following requirements:

Limitation #1: Required RE sites:
Restriction enzyme digestion is required for pre-treatment of DNA template. The absence of a desired restriction site within the PCR region leads to a failed walking reaction. Usually, multiple digestions have to be carried out to eliminate this problem. Applications that use this method (such as inverse PCR (IPCR), T-Linker PCR, Alu-PCR and other ligation-mediated PCR (LM-PCR)), as well kits developed from this method (PCR genome walker kit from Clontech, Genome walking kit from TaKaRa, and Vectorette or Splinkerette PCR genome walking kit from Sigma), all share this common problem.

Limitation #2: Required binding between arbitrary walking primers and the targeted DNA molecule:<
Arbitrary walking primers are usually degenerate. Such primers allow complementarily attachment to the targeted DNA molecule in order to initiate amplification under low stringency PCR conditions. The use of degenerate primers and low stringency PCR inevitably produce non-specific products. The absence of a complementary match between walking primers and the targeted DNA molecule leads to the failure of specific amplification. Hence, multiple walking primers have to be attempted. Kits, such as the DNA WalkingTM kit from Seegene, and certain PCR methods, such as randomly primed PCR (RP-PCR) and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR), all share this problem.

APA advantage:
Our new APA PCR walking method does not require restriction digestion or the attachment of multiple walking primers to the targeted region. Also, it takes advantage of immobilization and long-distance PCR. Therefore, it can produce longer walking fragments. Additionally, we have developed a cloning method that allows the cloning of specific fragments. See Figure 1 for details.

Figure 1: APA walking technology

Sample requirements


We accept genomic DNA, cDNA or plasmid DNA (such as BAC/PAC). Please provide a minimum of 20ug of DNA per sample in TE buffer. If possible, please provide a gel picture/sample confirming quality. Please send the material at room temperature via the courier of your choice to:

Bio S&T Inc.
 Attn: Areti Karadimos
 5020 Fairway Street, Suite 220
 Montreal (Quebec) H8T 1B8, Canada
 Tel: (514) 633-6006

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(s).

Starting sequences


We require150 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).

  • 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).

For further details or a quote, please contact Areti Karadimos at areti@biost.com