Molecular Cloning and Next-Generation-Sequencing Enzymes
The main purpose of molecular cloning is to insert the gene-of-interest (GOI) into a plasmid vector that facilitates cloning, clone selection and protein expression. Restriction enzymes and ligase often use to insert the GOI in-frame within the expression vector for subsequent protein expression.
Next-generation sequencing (NGS) technologies allow for massive parallel picoliter-scale amplification and detection of individual DNA molecules to generate hundreds of thousands of reads, providing the potential to reduce the time and complexity for DNA sequencing without the need for cloning.
G-biosciences NGS enzymes are uniquely optimized for end-repair (T4 DNA Polymerase and T4 Polynucleotide Kinase), dA-tailing (Taq DNA Polymerase), adaptor ligation (T4 DNA Ligase) and PCR amplification (high fidelity DNA Polymerase).
T4 DNA Polymerase catalyzes the synthesis of DNA in the 5´→ 3´ direction with the presence of template and primer. It has both DNA-dependent DNA polymerase activity and 3´→ 5´ exonuclease activity which make the enzyme useful for several molecular biology applications.
T4 Polynucleotide Kinase (PNK) exhibits 5’ polynucleotide kinase and 3’ phosphatase activity. It catalyzes 5’-phosphorylation of DNA and oligonucleotides via transferring the terminal phosphate of ATP to a 5’ hydroxyl group of a nucleic acid.
Taq DNA Polymerase possesses a 5´→3´ polymerase activity and a 5´-flap endonuclease activity. For A-tailing NGS sample, it is used to add an "A" base to the 3´ end of a blunt phosphorylated DNA fragment.
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between neighboring 5’-phosphate and 3’-hydroxyl termini in duplex DNA or RNA. It can be used to join DNA fragments with both sticky ends and blunt ends, and to repair nicks in double-stranded DNA. T4 DNA ligase covalently links the adapter to the DNA fragments, making a complete NGS library molecule.
High fidelity DNA Polymerase possesses 5´→3´ polymerase and 3´→5´ exonuclease (proofreading) activities. This unique Polymerase amplifies DNA with high processivity in highly TA- or GC-rich regions.