DNA Ligation Process and Ligases

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Questions and Answers

What is the primary feature of Taq polymerase that facilitates TA cloning?

5'-3' proofreading activity

Ability to add adenosine triphosphate to 5' ends

Its capacity to remove extended bases

Terminal transferase activity to add 3'-A overhang (correct)

Why might blunt-end ligation of PCR fragments into vectors be inefficient?

PCR products are too short

Vectors are always linearized

PCR fragments lack the necessary nucleotides

Blunt ends do not hybridize well (correct)

What type of cloning vector is specifically designed for use with TA cloning?

C-vector

T-vector (correct)

P-vector

R-vector

Which statement correctly describes the result of mixing PCR products with T-vectors?

A recombinant DNA is formed through DNA ligase Signup and view all the answers

How do polymerases such as Taq contribute to the PCR product's suitability for TA cloning?

By creating 3'-A overhangs at the ends of double-stranded products Signup and view all the answers

What is the primary role of DNA ligase in recombinant DNA technology?

To catalyze the joining of vector and DNA Signup and view all the answers

Which type of ligase is most commonly used in laboratories for DNA ligation?

T4 DNA ligase Signup and view all the answers

What cofactor is absolutely required for the activity of T4 DNA ligase?

ATP Signup and view all the answers

Which type of DNA ligase is specifically responsible for sealing DNA during nucleotide excision repair in mammals?

DNA ligase III Signup and view all the answers

How does E.coli DNA ligase perform ligation on blunt-ended DNA?

It requires high concentrations of polyethylene glycol to ligate blunt ends. Signup and view all the answers

What is a limitation of E.coli DNA ligase compared to T4 DNA ligase?

E.coli ligase is less versatile in the types of DNA it can ligate. Signup and view all the answers

Which ligase in mammals particularly helps ligate the nascent DNA of the lagging strand?

DNA ligase I Signup and view all the answers

Which type of nucleic acids can T4 DNA ligase effectively join?

RNA and RNA-DNA hybrids Signup and view all the answers

What is the purpose of using adaptors in DNA ligation?

To produce a molecule similar to a linker. Signup and view all the answers

How does homopolymer tailing contribute to the formation of sticky ends?

By producing a uniform tail of one type of nucleotide. Signup and view all the answers

What enzyme is specifically used for adding nucleotides during homopolymer tailing?

Terminal deoxynucleotidyl transferase. Signup and view all the answers

What is required for two tailed DNA molecules to ligate together?

The homopolymers must be complementary. Signup and view all the answers

What is the role of Klenow polymerase in the repair process after ligation?

To fill in nicks in the recombinant molecules. Signup and view all the answers

Which of the following is a characteristic of polydeoxyguanosine?

It is a type of homopolymer made entirely of guanine. Signup and view all the answers

How are nicks and discontinuities addressed after creating recombinant molecules?

By using two separate enzymes sequentially. Signup and view all the answers

What happens if complementary homopolymer tails are longer than 20 nucleotides?

They may form stable base-paired associations. Signup and view all the answers

What is the purpose of ligating an adaptor to a blunt-ended DNA fragment?

To create a new molecule with sticky ends Signup and view all the answers

What happens to the ends of adaptor molecules that could lead to dimer formation?

They base pair with each other Signup and view all the answers

What is necessary for a recombinant DNA molecule to be effectively utilized in a host cell?

It must have a base pairing but not be fully ligated. Signup and view all the answers

Why can't DNA ligase create a phosphodiester bridge between the adaptor molecule's ends?

The 5′ terminus lacks a phosphate group Signup and view all the answers

Which enzyme is primarily responsible for the blunt end ligation process described?

DNA topoisomerase Signup and view all the answers

What modification does the 5′-P terminus of the adaptor undergo after attachment?

It is converted back to a natural 5′-P form Signup and view all the answers

What role do DNA topoisomerases play during DNA replication?

They assist in unwinding the double helix. Signup and view all the answers

How do DNA topoisomerases achieve the separation of DNA strands?

By causing transient breaks in the backbone. Signup and view all the answers

How do adaptor molecules ensure that they do not ligate to themselves?

Their 5′ ends lack a phosphate group Signup and view all the answers

What specific sequence does the vaccinia topoisomerase cut to linearize a plasmid?

CCCTT Signup and view all the answers

What is a common result of base pairing between sticky ends of adaptor molecules?

Formation of stable dimers Signup and view all the answers

What happens to the DNA topoisomerase after it cuts the plasmid?

It remains covalently bound to the resulting blunt ends. Signup and view all the answers

Which feature distinguishes the 3′ terminus of the sticky end from typical DNA?

It carries a hydroxyl group Signup and view all the answers

What must occur before the prepared adaptor can function appropriately in DNA ligation?

Conversion of the 5′-OH terminus to a 5′-P form Signup and view all the answers

Which process is NOT a function of DNA topoisomerases?

Synthesizing RNA from DNA templates. Signup and view all the answers

What is the significance of homopolymer tailing in recombinant DNA construction?

It involves the synthesis of a homopolymer tail and ensures compatibility with a tailed vector. Signup and view all the answers

Study Notes

DNA Ligation

DNA Ligation is the process of joining DNA molecules together.
DNA ligase is the enzyme that catalyzes the reaction.
It is the final step in constructing recombinant DNA molecules.

Types of Ligases

E. Coli DNA Ligase:
- Uses energy from cleaving NAD to create a phosphodiester bond.
- Cannot ligate blunt-ended DNA efficiently, except under conditions of molecular crowding with polyethylene glycol.
- Cannot efficiently join RNA to DNA.
T4 DNA Ligase:
- Most commonly used ligase in laboratories.
- Ligates cohesive ends of DNA, oligonucleotides, RNA, and RNA-DNA hybrids but not single-stranded nucleic acids.
- Ligates blunt-ended DNA more efficiently than E. Coli DNA Ligase.
- Requires ATP as a cofactor.
Mammalian Ligases:
- Include DNA ligase I, III, and IV.
- DNA ligase I ligates the nascent DNA of the lagging strand, after Ribonuclease H removes the RNA primer from Okazaki fragments.
- DNA ligase III complexes with DNA repair protein XRCC1 to aid in sealing DNA during nucleotide excision repair and recombinant fragments.
- DNA ligase IV complexes with XRCC4.

Adaptors

Adaptors are synthesized to have one sticky end and one blunt end.
The sticky end of the adaptor is modified to have a 5′-OH terminus, instead of a 5′-P terminus.
This modification prevents the adaptors from ligating to themselves – they can only ligate to blunt-ended DNA molecules.
The abnormal 5′-OH terminus is converted to a natural 5′-P form by treatment with polynucleotide kinase, producing a sticky-ended fragment for insertion into a vector.

Homopolymer Tailing

A technique used to produce sticky ends on blunt-ended DNA molecules.
Uses terminal deoxynucleotidyl transferase to add a series of nucleotides onto the 3′-OH termini of a double-stranded DNA molecule.
By adding only one deoxyribonucleotide, a homopolymer tail is constructed.
Complementary homopolymers are required for ligation.
For example, the vector might have a poly(dC) tail, and the DNA to be cloned a poly(dG) tail.
Base pairing between the homopolymers occurs when the molecules are mixed.
The tails are not always the same length, so the base-paired recombinant molecules may have nicks and discontinuities.
Klenow polymerase fills in the nicks, while DNA ligase creates the phosphodiester bonds, completing the repair.
If the homopolymer tails are >20 nucleotides, the recombinant DNA molecule is stable enough to be introduced into the host cell.
The host cell's own DNA polymerase and ligase repair the remaining inconsistencies.

Blunt End Ligation with DNA Topoisomerase

A more efficient method for blunt end ligation.
Uses a special type of cloning vector linearized by the nuclease activity of a topoisomerase.
Topoisomerases remove or add turns in the double helix, allowing DNA replication and supercoiling.
They separate the two strands of DNA without rotating the double helix.
Topoisomerases have both nuclease and ligase activities.
Vector linearization is achieved with vaccinia virus DNA topoisomerase.
Topoisomerase enzymes remain covalently bound to the resulting blunt ends, allowing for vector storage.
Pfu DNA polymerase can be used to remove extended bases.

TA Cloning

Popular method for cloning PCR products amplified using Taq polymerase or other polymerases lacking 5'-3' proofreading activity.
These polymerases add a single 3'-A overhang to each end of the double-stranded PCR product.
PCR products with 3'-A overhangs can be directly cloned into linearized cloning vectors with complementary single base 3'-T overhangs.
The vectors are called T-vectors.
Complementary overhangs hybridize and the recombinant DNA is formed by DNA ligase.

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Description

Explore the crucial process of DNA ligation, which involves joining DNA molecules together through the action of ligases. This quiz covers different types of ligases, including E. Coli, T4, and mammalian ligases, highlighting their functions and applications in recombinant DNA technology.