What is DNA end repair?

The End-It DNA End-Repair Kit is used to convert DNA with damaged or incompatible 5′-protruding and/or 3′-protruding ends to 5′-phosphorylated, blunt-end DNA for fast and efficient blunt- end ligation into plasmid, cosmid, fosmid, BAC, other cloning vectors, or next- gen DNA sequencing adaptors.

How are blunt ends ligated?

Blunt End Ligation They cut the desired DNA portion. Usually, a straight cut creates blunt ends or non-overhanging ends. These ends can be joined using a DNA ligase enzyme. The joining of two blunt ends is called blunt end ligation.

Why is blunt-end ligation important?

The attaching of blunt-ended DNA fragments by the enzyme DNA ligase is known as blunt end ligation. This is a crucial laboratory procedure used in the molecular cloning of DNA. During the process the linearized plasmid vector and the blunt-ended insert are mixed with DNA ligase.

What is end repair enzyme?

The End Repair Enzyme Mix contains an optimized mixture of T4 DNA Polymerase and Klenow Fragment to achieve highly effective blunting of fragmented DNA, and T4 Polynucleotide Kinase for efficient phosphorylation of DNA ends. The 10X End Repair Reaction Mix contains optimized reaction buffer, ATP and dNTPs.

What is the purpose of end repair?

The end repair process removes the 3′ overhangs with Klenow fragment and fills in the 5′ overhangs with a T4 DNA polymerase. The end repair cocktail also contains T4 polunucleotide kinase (PNK) which phosphorylates the 5′ ends and ensures the 3′ ends carry a hydroxyl group.

What is end repair and a tailing?

After fragmentation, the DNA fragments are end repaired or end polished. Generally, a single adenine base is added to form an overhang via an A-tailing reaction. This A overhang allows adapters containing a single thymine overhanging base to base pair with the DNA fragments.

Can DNA ligase joins blunt ends?

Ligases can join any DNA fragments with ‘blunt’ ends. They can also join DNA fragments with ‘sticky’ ends, but only if the nucleotides on the strands are complementary. To get complementary ‘sticky ends’ the DNA fragments to be joined must be cut with the same restriction enzyme.

Can blunt ends be ligated to sticky-end?

Traditionally what would be done in this case is to blunt the sticky ends with a polymerase (usually T4 polymerase) and do a blunt end ligation. In this case you will have to do some screening to find clones with the desired orientation since it can ligate in either direction.

What is da tailing?

Tailing is an enzymatic method for adding a non-templated nucleotide to the 3′ end of a blunt, double-stranded DNA molecule. Tailing is typically done to prepare a T-vector for use in TA cloning or to A-tail a PCR product produced by a high-fidelity polymerase (not Taq) for use in TA cloning.

What is the purpose of the end repair procedure in next generation sequencing?

Because DNA fragmentation does not result in homogeneous, blunt-ended fragments, end repair is needed to ensure that each molecule is free of overhangs, and contains 5′ phosphate and 3′ hydroxyl groups.

What is NGS end repair?

The End-Repair mix converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′- phosphorylated, blunt-ended DNA. This high-concentration formulation of the End-Repair Mix is compatible with applications requiring >1 microgram of DNA to be prepared for blunt-end ligation.

What is a tailing sequencing?

Can blunt ends be ligated?

Blunt end ligation does not involve base-pairing of the protruding ends, so any blunt end may be ligated to another blunt end. Blunt ends may be generated by restriction enzymes such as SmaI and EcoRV.

Can t4 ligase Ligate blunt end?

Catalyzes the formation of a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA and some DNA/RNA hybrids (1).

Which restriction enzymes produce blunt ends?

So, the correct answer is ‘Eco RV’.

What is a blunt end cut?

The simplest DNA end of a double stranded molecule is called a blunt end. Blunt ends are also known as non-cohesive ends. In a blunt-ended molecule, both strands terminate in a base pair.

How blunt ends are formed?

The enzyme cuts between two bases directly opposite each other on the complementary strands of the double stranded DNA. This cleavage results in the formation of blunt ended DNA fragments. Blunt Ends are made commonly at a specific site when trying to make a piece of recombinant homologous DNA.

How do you make blunt DNA ends?

You can create blunt ends by filling in single stranded overhangs remaining after physically shearing (see Fig. 2) or cutting with restriction endonucleases that generate sticky ends. The single-stranded overhangs can be repaired using a mixture of DNA polymerases such as T4 polymerase and the Klenow fragment.

Does PCR end repair make blunt end?

I checked the function of end repair, if I’m not wrong, end repair will make blunt end and add hydroxy to 3′, phosphate to 5′. But if it’s PCR product, will it have this itself? I mead I don’t shear the DNA. I use gel purification or PCR purification after PCR reaction.

Can blunt end inserts be directionally cloned?

Blunt ends are universally compatible with other blunt ends, so the directionality you can achieve by using sticky ends with different overhang sequences is not possible. So, what if you are hoping for a directionally cloned insert?

How does blunt-end cloning work?

As mentioned above in describing how blunt-end cloning works, a 5’-phosphate group is covalently linked to the corresponding 3’-hydroxyl group of the adjoining base. This happens on both complementary strands and for that reason a blunt ended insert could be ligated into the vector in two orientations (i.e. in both directions).