Molecular Biology: Restriction Enzymes and Cloning

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Questions and Answers

Which class of polymerase uses RNA as a template to synthesize RNA?

DNA dependent RNA polymerase

RNA dependent RNA polymerase (correct)

DNA dependent DNA polymerase

Reverse transcriptase

What is one function of the 3’–>5’ proof-reading activity of DNA polymerases?

To extend an existing nucleic acid strand

To remove RNA primers from the lagging strand (correct)

To initiate the replication process

To synthesize DNA from RNA templates

What is the primary reason the Klenow fragment is used in molecular biology?

For its 5’–>3’ exonuclease activity

For its intrinsic specificity for substrates

To initiate the synthesis of RNA

For synthesizing double-stranded DNA from single-stranded templates (correct)

Which of the following polymerases lacks intrinsic specificity for their nucleic acid substrate?

Terminal deoxynucleotidyl transferase Signup and view all the answers

What does a RNA dependent DNA polymerase use as a template for synthesis?

RNA Signup and view all the answers

What is the main function of type II restriction enzymes?

To recognize and cut specific palindromic sequences Signup and view all the answers

What do type II restriction enzymes typically produce when cleaving DNA?

Sticky ends and blunt ends Signup and view all the answers

How many type II restriction enzymes have been discovered?

Over 400 Signup and view all the answers

What is usually required for two DNA molecules to be joined together?

Compatible ends Signup and view all the answers

What technique can be used to determine the positions of restriction enzyme cut sites on a DNA molecule?

Restriction mapping Signup and view all the answers

What is indicated by the term 'sticky ends' in the context of DNA cleavage?

3' or 5' ends that can easily hybridize with other DNA fragments Signup and view all the answers

What aspect determines the specificity of type II restriction enzymes?

The recognition sequence length of 4-8 base pairs Signup and view all the answers

How do researchers typically generate restriction maps if they know the DNA sequence?

By employing software to find restriction enzyme sequences Signup and view all the answers

What is one of the main advantages of using Taq polymerase in PCR?

It is isolated from a thermophilic bacterium. Signup and view all the answers

Which feature is characteristic of Taq polymerase?

5’-3’ exonuclease activity Signup and view all the answers

What is a primary drawback of using Taq polymerase for PCR?

Added 3' overhangs of a single 'A'. Signup and view all the answers

Which polymerase offers a significant improvement over Taq polymerase due to its proof-reading activity?

Pfu polymerase Signup and view all the answers

What is a notable characteristic of Pfu polymerase?

It has ten-fold lower error rate than Taq polymerase. Signup and view all the answers

Under what conditions does Taq polymerase exhibit its optimal activity?

75–80 °C Signup and view all the answers

What challenge does Taq polymerase face when dealing with GC-rich DNA?

Poor thermotolerance at high temperatures. Signup and view all the answers

How has the market responded to the drawbacks of Taq polymerase?

By developing polymerases with improved error rates. Signup and view all the answers

What is the primary function of DNA ligase in molecular biology?

To catalyze the formation of phosphodiester bonds Signup and view all the answers

Why are blunt end ligations considered to be much less efficient than sticky end ligations?

They have less specific binding. Signup and view all the answers

What is the role of alkaline phosphatases in DNA manipulation?

To prevent self-ligation of vector DNA Signup and view all the answers

Which enzyme is responsible for adding a 5’-phosphate to the ends of DNA?

DNA polynucleotide kinase Signup and view all the answers

What might be a consequence of using a DNA phosphatase on a DNA vector?

Prevents self-ligation Signup and view all the answers

How does DNA polynucleotide kinase facilitate ligation in DNA experiments?

By adding a 5’-phosphate to the DNA ends Signup and view all the answers

What specific form of energy is required for the function of DNA ligase?

ATP or NAD+ Signup and view all the answers

Which of the following statements correctly describes the addition of a 5’-phosphate to DNA via PNK?

PNK adds phosphate from ATP to the 5’ end of DNA. Signup and view all the answers

What is a primary characteristic of Terminal deoxynucleotidyl transferase (TdT)?

It functions without requiring a template for nucleotide addition. Signup and view all the answers

Which of the following accurately describes the fidelity of Phusion high-fidelity DNA polymerase compared to Taq and Pfu?

Phusion is 52x more accurate than Taq. Signup and view all the answers

What type of enzyme are reverse transcriptases (RT) primarily classified as?

RNA-directed DNA polymerases. Signup and view all the answers

Which of the following is a common use for eubacterial-type RNA polymerases?

Regulated transcription in vivo from lac promoters. Signup and view all the answers

What is one consideration when selecting a reverse transcriptase enzyme?

Fidelity. Signup and view all the answers

What is the synthesis rate of Phusion high-fidelity DNA polymerase?

1 kb every 15–30 seconds. Signup and view all the answers

Which option describes a use of reverse transcriptases in molecular biology?

Creation of cDNA for RT-PCR. Signup and view all the answers

What does the sigma factor do in eubacterial-type RNA polymerases?

It recognizes the promoter for transcription initiation. Signup and view all the answers

Study Notes

Restriction Enzymes

Restriction enzymes are bacterial enzymes that cut specific palindromic DNA sequences.
They are homodimers and can recognize 4-8 base pair sequences
They cleave sugar-phosphate backbones leaving a 5’ phosphate, 3’ overhang, 5’ overhang (sticky ends) or a blunt end.

Cloning

Two DNA molecules can be joined if the ends are compatible.
Example of joining is cloning DNA into a plasmid.

Restriction Maps

Using different combinations of restriction enzymes allows us to map their approximate positions on a DNA molecule.
Alternatively, we can use software to search for the restriction enzyme sequences in a known DNA sequence to generate a restriction map.

Restriction Fragment Length Polymorphism

Restriction Fragment Length Polymorphism (RFLP) analysis identifies variations in DNA fragment lengths due to different restriction site patterns amongst individuals.

DNA Ligase

DNA ligases catalyze the formation of a phosphodiester bond in double-stranded DNA.
This bond forms between the 5’ phosphate and 3’ OH of adjacent nucleotides.
Requires ATP or NAD+ as energy source.
Ligation of blunt ends is less efficient.

DNA Phosphatase

Alkaline phosphatases, such as calf intestinal phosphatase, remove 5’ phosphate from DNA or RNA.
Used to prevent self-ligation of vector DNA.

DNA Polynucleotide Kinase

PNK adds 5’ phosphate from ATP to the 5’ end of double-stranded or single-stranded DNA.
Used to label DNA with 32P or to allow ligation (e.g. of PCR products).

Polymerases

Polymerases make nucleic acid polymers
Use nucleotides as building blocks, and an existing nucleic acid strand as a template for copying
Divided into four classes:
- DNA dependent DNA polymerase (DNA polymerases)
- DNA dependent RNA polymerase (RNA polymerases)
- RNA dependent DNA polymerase (Reverse transcriptases)
- RNA dependent RNA polymerase (RNA replicases)
There are other types of DNA or RNA polymerases that extend an existing strand such as Terminal deoxynucleotidyl transferase (TdT) or poly(A) polymerase.

DNA Polymerases

E.coli DNA Polymerase I was the first DNA polymerase used in molecular biology.
Has three different activities:
- 5’ to 3’ polymerase activity
- 3’ to 5’ proof reading activity
- 5’ to 3’ exonuclease activity (RNA primer removal on lagging strand)
The Klenow fragment is a modified version of DNA Polymerase I which lacks the 5’ to 3’ exonuclease activity.
The Klenow fragment is used for synthesis of dsDNA from single-stranded templates, filling in recessed 3’ ends, digesting protruding 3’ overhangs and preparation of radioactive DNA probes.

Thermostable DNA Polymerases

PCR methods originally used Klenow as the DNA polymerase.
Major advance was the discovery of thermostable polymerase, Taq polymerase, isolated from thermophilic bacteria, Thermus aquaticus
Taq polymerase has an optimum activity between 75-80 °C, with a half-life of >40 mins at 95 °C.
Taq polymerase exhibits similar structure to DNA pol I, but lacks a functional proof-reading domain, and degraded DNA primers.
Taq polymerase is used for PCR due to its stability and cheap cost.

Drawbacks of Taq Polymerase

Lack of proof-reading results in higher error rates during strand synthesis and addition of 3’ overhangs of a single ‘A’ to PCR products
Thermotolerance is poor at very high temperatures needed to denature GC-rich DNA

Pfu Polymerase

Pfu Polymerase is from the hyperthermophilic archaeon Pyrococcus furiosus
It is a two-subunit enzyme with high thermo-tolerance and proof-reading activity with a 10-fold lower error-rate than Taq polymerase.
Pfu Polymerase is slower than Taq Polymerase.

Phusion Polymerase

Phusion Polymerase is a novel Pyrococcus-like enzyme fused with a processivity-enhancing domain
It shows increased fidelity and high synthesis rate.

Terminal Deoxynucleotidyl Transferase (TdT)

Eukaryotic enzyme that adds random bases to the 3’ end of single- or double-stranded DNA
Does not require a template, just a 3’ OH group
Used for homopolymer tailing, labeling of the 3’ ends of DNA with modified nucleotides, and ‘biological’ oligonucleotide synthesis.

RNA Polymerases

Eubacterial type RNA polymerase (e.g.E.coli RNAP) is a multi-subunit complex with a sigma factor for promoter recognition.
It is used for regulated transcription in vivo from the lac promoter and transcription in vitro from E.coli promoters.
Bacteriophage-type RNA polymerase (e.g.T7, T3 & SP6 RNAP) is a single subunit enzyme with promoter recognition and RNA synthesis activities.
It has high specificity for the promoter and high fidelity.
It is used for regulated transcription in vivo from the phage promoter and transcription in vitro.

Reverse Transcriptases (RT)

RNA directed DNA polymerases from retroviruses.
Use RNA or SS DNA as a template to make a complementary DNA strand (cDNA).
Requires a 3’ OH group to extend an existing strand.
Two commonly used RT enzymes are the Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukaemia Virus (MMLV) Reverse Transcriptase.
Used for cDNA synthesis, RT-PCR.
Considerations when choosing an RT include: Fidelity, Thermostability and processivity.

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Description

This quiz explores the fundamentals of restriction enzymes, their role in DNA manipulation, and the process of cloning. Additionally, it covers the creation of restriction maps and the analysis of DNA variations through Restriction Fragment Length Polymorphism (RFLP). Test your knowledge on how these tools are essential for genetic engineering.