Features and Benefits
The Universal Vectorette System offers the flexibility to generate Vectorette libraries from purified genomic DNA by BamHI, ClaI, EcoRI, HindIII or blunt restriction enzyme digests. This system can be used with 1ug template genomic DNA or less, and provides a time saving alternative to traditional library construction and screening. The protocol can also be modified for high throughput applications.
Sequencing of yeast artificial chromosome (YAC) termini
Sequencing of cosmid insert termini
Mapping of promoters, introns, microsatellites, SSR′s and STR′s
Sequencing of large clones without sub-cloning
Mapping of regions containing deletions, insertions and translocations
Gap-filling in genome mapping projects
Identification of flanking genomic sequences of transgenes in transgenic organisms
- Cell-free gene manipulation replaces cloning and subcloning in many molecular genetics projects
- Generate results from the two or three-step procedure in a single day
- Amplify up to 6 kb from genomic DNA with high fidelity and specificity
- Eliminates the need for nested PCR in most applications
The Vectorette system is a PCR-based method for DNA walking and mapping that uses a form of unidirectional PCR for amplifying and sequencing unknown genomic or large construct DNA. The system eliminates the time-consuming need to make and screen libraries to obtain overlapping clones that use conventional nucleic acid purification and screening procedures. A Vectorette unit is employed, which consists of a double-stranded linker with an internal mismatched region and a sticky end.
The Universal Vectorette system uses three simple steps to obtain DNA sequence information:
Step 1: Genomic or large construct DNA containing target sequence is digested with a restriction enzyme and ligated to a Vectorette unit to create a Vectorette library. The Vectorette library consists of DNA fragments that have a Vectorette unit on each end.
Step 2: PCR is performed on the Vectorette library using a primer complementary to the mismatched region of the Vectorette unit (Vectorette primer provided) and a specific primer to known DNA sequence. In the first PCR cycle, primer extension occurs only from the specific PCR primer that hybridizes to the known sequence in the DNA fragment within the Vectorette library. Extension from this primer generates a unique sequence as the polymerase reads through the mismatched portion of the Vectorette. Subsequent PCR cycles generate a DNA fragment between the known sequence and the Vectorette unit on the end of the fragment. Any Vectorette fragment that does not contain a sequence that is complementary to the specific primer will not generate a PCR product.
Step 3: A separate sequencing primer is included (slightly nested) that can be used to perform a sequencing reaction from the Vectorette end. PCR products are typically obtained from a single PCR run, however, nested primers are included to increase specificity when amplifying more complex templates. The PCR products generated by the Vectorette system can be used directly for cycle sequencing or cloned into commercially available vectors for further characterization.
Vectorette is a trademark of Sigma-Aldrich Co. LLC