JBS DNA-Shuffling Kit

Description: JBS Mutagenesis Series Within three billion years of evolution, nature has produced a plethora of proteins simply by repeated cycles of random mutagenesis followed by in vivo selection for superior function of the encoded proteins.

This example of natural evolution has guided researchers within the last two decades to develop strategies for in vitro permutation of proteins. Among the variety of strategies applied, three major powerful techniques have emerged.

→ JBS dNTP-Mutagenesis Kit #PP-101

The rate of mutagenesis achieved by error-prone PCR is in the range of 0.6-2.0%. → JBS Error-Prone Kit #PP-102

This method allows the recombination of sequences from different, related genes. The overall rate of mutagenesis is approx. 0.7%. → JBS DNA-Shuffling Kit #PP-103

Jena Bioscience now offers all components necessary for each of these techniques 'ready-to-go' in a separate kit, accompanied by a streamlined documentation that maximizes success.

Content

DNase I (yellow cap)

0.1 units/μl, 100 μl

Digestion Buffer (blue cap)

10x concentration, 100 μl

DNase Stop Solution (yellow cap)

100 μl

Taq Polymerase (red cap)

5 units/μl, 40 μl

Shuffling Buffer (green cap)

10x concentration, 200 μl

dNTP Mix (white cap)

10 mM each dNTP (dATP, dCTP, dGTP,dTTP), 40 μl

PCR-grade Water (white cap)

3x 1 ml

DNA shuffling can be divided into single gene shuffling and multiple gene shuffling (Fig. 1).

In single gene shuffling only one gene is digested and subsequently reassembled resulting in point mutations at a rate of approx.

0.7%.

The major application of DNA shuffling in protein evolution is multiple gene shuffling (often referred to as molecular breeding). In this technique several homologous DNA sequences are digested and subsequently reassembled.

The result is a library of chimaeric genes containing additional point mutations.

A pivotal step in DNA shuffling is the digest of the gene of interest for production of fragments of appropriate size. Therefore, all reagents in the Kit are optimised to obtain fragments of the desired size within a convenient time frame (Fig.

2).

Usually best results were achieved using fragments with mean sizes of 70-280 bp. Note that a complete DNase I digest results in very short fragments that cannot be amplified by subsequent PCR.

Recommended assay preparation

1. DNase I digest of gene(s) of interest

• For a total volume of 50 μl, use 5 μl of 10x Digestion Buffer in an appropriate vial and add 0.5-2 μg of each starting DNA
• Add PCR-grade Water to a final volume of 50 μl
• Add 0.1 u (1.0 μl) DNase I per μg of starting DNA
• Depending on the desired fragment size incubate at 37°C for up to 8 min (refer to Fig. 2)
• Quench the digest by addition of 5 μl DNase Stop Solution followed by heat inactivation of the DNase I at 75°C for 10 min.
• Isolate fragments of the desired size range by agarose gel electrophoresis and purify using standard procedures.

2. First PCR (without primers)

• For a 50 μl reaction, take 5 μl of the 10x Shuffling Buffer in an appropriate vial and add the purified DNA fragments from step 1 to a final concentration of 10-20 ng/μl
• Add 1 μl of dNTP Mix
• Add 2.5 units (0.5 μl) of Taq Polymerase
• Add PCR-grade Water to a final volume of 50 μl
• Recommended thermocycling conditions: 1. Denaturation: 94°C 90 sec 2. Annealing: 55°C 30 sec 3. Extension: 72°C 30 sec Number of cycles: 30-45
• Purify PCR product using standard procedures

3. Second PCR (with primers)

• For a 50 μl reaction, take 5 μl of the 10x Shuffling Buffer and add 2 μl of the PCR product from step 2
• Add 1 μl of dNTP Mix
• Add primers to a concentration of 0.8 μM each
• Add 2.5 units (0.5 μl) of Taq Polymerase
• Add PCR-grade Water to a final volume of 50 μl
• Recommended thermocycling conditions: 1. Denaturation: 94°C 30 sec 2. Annealing: 55°C 30 sec 3. Extension: 72°C 30 sec Number of cycles: 15