As a third commercial application of Seegene's proprietary ACP (Annealing Control Primer) Technology maximising PCR specificity, the DNA Walking SpeedUp™ Kit is directed to methods of using our unique DNA Walking ACP (DW-ACP) primer which is designed to capture unknown target sites and the optimised PCR conditions (referred to as DW ACP-PCR™ Technology hereunder). Due to the unique features of the DW-ACP primer system, DW ACP-PCR™ Technology enables the researchers to obtain only genuine unknown target products up to the length (up to 2 Kb) of which Taq polymerase is able to synthesise. This method provides the most powerful and revolutionary way to directly amplify unknown sequences adjacent to known sequences (Fig. 1). Whole genomic DNA, total RNA, cDNA or plasmid (clone) can be used as starting material.

The kit provides four different DW-ACP 1, 2, 3, 4 each having "GGTC" sequence at 3' end which is present in every 1-2 kb template on average. To extend this, unknown target sequences can be amplified using DW-ACPs and TSPs (Target Specific Primers).

Fig. 1. DNA Walking ACP-PCR™ Technology.
A. Flow chart of DNA Walking ACP-PCR™. DW-ACP: DNA Walking-Annealing Control Primer. Four DW-ACPs are supplied and each requires an individual PCR. TSP1: Target Specific Primer; TSP2: First Nested Target Specific Primer; TSP3: Second Nested Target Specific Primer;
B. Results of control PCR. 1: DW-ACP1; 2: DW-ACP2; 3: DW-ACP3; 4: DW-ACP4

Features

• The simplest method
  This method requires NO complex process, NO restriction enzyme digestion, NO immobilisation, and NO complex PCR.

• The fastest method
  Within 1 day, products are able to be obtained at least tens times faster than with currently available methods; NO library construction, NO library screening, and NO subcloning.

• The most reliable method
  Only target products are able to be obtained so NO false products are allowed.

Within ONE day,
- Up to 1-2 kb can be sequenced by each DNA walking
- Up to 1-2 kb promoter region can be cloned
- Transgene location can be determined
- Deletion or insertion can be determined
- Splicing form can be determined

Components

1. 2.5µM DW-ACP1 primer for first PCR reaction
DW-ACP1: 5'-ACP-AGGTC-3'
2. 2.5µM DW-ACP2 primer for first PCR reaction
DW-ACP2: 5'-ACP-TGGTC-3'
3. 2.5µM DW-ACP3 primer for first PCR reaction
DW-ACP3: 5'-ACP-GGGTC-3'
4. 2.5µM DW-ACP4 primer for first PCR reaction
DW-ACP4: 5'-ACP-CGGTC-3'
5. 10µM DW-ACP-N primer for second PCR reaction
DW-ACP-N: 5'-ACPN-GGTC-3'
6. 10µM Universal primer for third PCR reaction
Uni-primer: 5'-TCACAGAAGTATGCCAAGCGA-3'
Taq polymerase
2mM dNTP

(* Each items are available for individual purchase.
Please contact us at biogene@biogene.com for details.)

Reagents and Equipment to be Supplied by User

Target specific primer (TSP), Thermal cycler, PCR purification kit

Storage Conditions

This product is shipped at ambient temperature. Store the template below -20°C for long term storage. Avoid multiple freeze-thaw cycles.