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HUMANVS Sigma

Vectorette Genomic Systems

Human Genomic Vectorette Library

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Properties

usage   1 kit sufficient for 20 reactions (50 μl reaction volume)
shipped in   wet ice
storage temp.   2-8°C

Description

Features and Benefits

• 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 Genomic Library is a preconstructed set of genomic human DNA digested with a specific restriction enzyme and ligated to a Vectorette unit on each end of the digest to create a Vectorette library. This provides added convenience by completing the first step of the Universal Vectorette system. The Vectorette Genomic Library has been constructed using four restriction enzymes: Bgl II, EcoR I, Hind III, and Pvu II (blunt end).

Ideal for:
Genome walking
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

Other Notes

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.

Principle

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.

Legal Information

Vectorette is a trademark of Sigma-Aldrich Co. LLC

Price and Availability

Protocols & Articles
Peer-Reviewed Papers
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