Biotechnology Tools: Enzymes and Ligation

Questions and Answers

What type of DNA molecules can DNA ligase catalyze during the joining reactions?

Only blunt-ended molecules

Neither blunt-ended nor sticky-ended molecules

Both blunt-ended and sticky-ended molecules (correct)

Only sticky-ended molecules

What is a common method to create sticky ends on blunt-ended DNA fragments?

Using single-stranded DNA

Employing polymerase chain reaction

Incorporating linkers (correct)

Using heat denaturation techniques

What characteristic do linkers possess that allows them to be attached by DNA ligase?

They are naturally occurring within cells

They have complementary sticky ends

They are produced exclusively in vivo

They contain known nucleotide sequences and a restriction site (correct)

During gene cloning, what is a potential issue when using a vector with sticky ends but blunt-ended DNA fragments?

It is difficult to ligate them without modifications Signup and view all the answers

What is the role of a common restriction site in the use of linkers?

It facilitates the recognition and cutting by restriction enzymes Signup and view all the answers

What is the primary purpose of using linkers in DNA modification?

To attach synthetic oligonucleotides at a high concentration Signup and view all the answers

What happens when a blunt-ended DNA molecule contains BamHI recognition sequences during the restriction step?

Both the linkers and the DNA molecule are cleaved Signup and view all the answers

What is one drawback of using linkers to attach sticky ends?

They may cut the original DNA fragment if it contains recognition sequences Signup and view all the answers

How do adaptors differ from linkers in their function?

Adaptors are designed to avoid cleavage issues faced by linkers Signup and view all the answers

What is produced after BamHI cleaves the chains at the recognition sequences?

Cleaved linkers and multiple DNA fragments Signup and view all the answers

What is required for two blunt-ended DNA molecules to ligate after the addition of adaptors?

That they both undergo a restriction step Signup and view all the answers

What characterizes a homopolymer?

It contains repeating units of the same nucleotides Signup and view all the answers

Why is it important for poly(dG) and poly(dC) tails to be complementary?

To ensure proper base pairing and ligation Signup and view all the answers

What is the first step in repairing nicks in recombinant DNA that has undergone homopolymer tailing?

Employing Klenow polymerase to fill in the nicks Signup and view all the answers

How does terminal deoxynucleotidyl transferase contribute to the creation of sticky ends?

It adds a series of identical nucleotides to the ends Signup and view all the answers

What might happen if the poly(dG) and poly(dC) tails are not the same length?

Nicks and discontinuities may occur in the resulting DNA Signup and view all the answers

What is the role of Klenow polymerase in the repair reaction following homopolymer tailing?

To fill in nicks within the recombinant DNA Signup and view all the answers

If the complementary homopolymer tails are longer than approximately 20 nucleotides, what is the outcome?

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

What is the primary role of DNA ligase in the process of constructing a recombinant DNA molecule?

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

Which ligase is most commonly used in laboratory settings for joining cohesive ends of DNA?

T4 DNA ligase Signup and view all the answers

What energy source does E.coli DNA ligase utilize to create the phosphodiester bond?

Nicotinamide adenine dinucleotide (NAD) Signup and view all the answers

Which of the following statements is true about T4 DNA ligase?

It ligates RNA-DNA hybrids. Signup and view all the answers

What is one of the functions of DNA ligase I in mammals?

To ligate DNA after RNA primer removal Signup and view all the answers

Which ligase is associated with DNA repair in mammals, particularly during nucleotide excision repair?

DNA ligase III Signup and view all the answers

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

It utilizes NAD for energy. Signup and view all the answers

Which of the following ligases can efficiently ligate blunt-ended DNA?

T4 DNA ligase Signup and view all the answers

What must be done to blunt-ended molecules generated by restriction enzymes before ligation with a vector?

They must be treated with alkaline phosphatase. Signup and view all the answers

What is the role of topoisomerases during the ligation process?

They repair the discontinuities in the DNA. Signup and view all the answers

Which property of Taq polymerase complicates the blunt-end ligation of PCR products?

It adds a 3′ A overhang to each end. Signup and view all the answers

What final structure is obtained after the topoisomerase ligates the 5′-OH and 3′-P ends?

A double-stranded molecule with two discontinuities. Signup and view all the answers

What happens to the phosphatased molecules when added to the vector?

They reactivate the bound topoisomerases. Signup and view all the answers

Which enzyme can be used to fill in the ends of blunt-ended DNA molecules for cloning after PCR?

Klenow polymerase. Signup and view all the answers

What is the primary focus when cloning blunt-ended molecules?

Converting ends from 5′-P to 5′-OH. Signup and view all the answers

What is a key characteristic of the ligation process involving blunt-ended molecules?

Only one strand is ligated at each junction point. Signup and view all the answers

Study Notes

Ligation and DNA Ligases

Ligation is the process of joining DNA molecules, essential for constructing recombinant DNA.
DNA ligase is the enzyme that catalyzes the ligation reaction.
Various types of ligases are utilized depending on the organisms and conditions.

Types of DNA Ligases

E. coli DNA Ligase:
- Encoded by the lig gene and uses NAD to create phosphodiester bonds.
- Limited in its ability to ligate blunt-ended DNA unless molecular crowding conditions are imposed.
T4 DNA Ligase:
- Commonly used in laboratories; ligates cohesive ends, oligonucleotides, RNA, and RNA-DNA hybrids.
- Requires ATP as a cofactor and is more efficient with blunt-ended DNA compared to E. coli ligase.
Mammalian Ligases:
- DNA Ligase I: Seals Okazaki fragments during lagging strand synthesis after RNA primer removal.
- DNA Ligase III: Works with XRCC1 in nucleotide excision repair and recombinant DNA.
- DNA Ligase IV: Functions in DNA double-strand break repair, complexes with XRCC4.

Methods for Generating Sticky Ends

Linkers:
- Short double-stranded DNA pieces with known sequences that add restriction sites to blunt-ended DNA.
- Digestion with restriction enzymes (e.g., BamHI) produces cohesive ends for ligation.
Adaptors:
- Similar to linkers but do not leave a modified blunt end after ligation.
- Designed to prevent recognition site issues, ensuring blunt-ended molecules are retained.
Homopolymer Tailing:
- Involves using terminal deoxynucleotidyl transferase to add nucleotide tails to blunt-ended DNA.
- Usually, poly(dG) is used on one molecule and poly(dC) on the other to facilitate ligation.

Challenges with Linkers and Adaptors

Linkers can inadvertently cleave associated blunt-ended DNA if recognition sites are present within the original DNA fragments.
Adaptors avoid this complication, allowing the blunt-ended DNA to remain intact until further processing.

Further Processing of Blunt Ends

Alkaline phosphatase treatment converts 5′-P ends to 5′-OH, enabling ligation.
Ligation using topoisomerase forms covalent bonds between the DNA strands, resulting in the formation of recombinant DNA.

Special Considerations for PCR Products

PCR amplification often leaves a single 3′ A overhang due to polymerase activity, necessitating blunting before ligation.
Klenow polymerase can be employed to fill in the ends, preparing PCR products for cloning compatibility.

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Description

Explore the essential tools of biotechnology, focusing on enzymes used in the ligation process to join DNA molecules. This quiz delves into the types of ligases and their functional importance in constructing recombinant DNA. Test your understanding of these critical concepts in molecular biology.