Instruction 8 - DNA Vectors and Restriction Enzymes

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Questions and Answers

What is the primary purpose of adding a specific antibiotic to agar plates when growing bacteria after plasmid transformation?

To inhibit the growth of bacteria that have not taken up a plasmid with a resistance gene. (correct)

To increase the mutation rate of the bacteria.

To directly identify the inserted gene via fluorescent tagging.

To accelerate the growth of all bacteria present.

What is the main goal when collecting bacterial clones from agar plates after plasmid transformation?

To evenly distribute bacteria for further amplification without plasmid extraction.

To isolate and identify plasmids containing the correctly inserted gene. (correct)

To lyse all bacteria and release all plasmids simultaneously.

To measure the overall bacterial growth rate in the presence of antibiotics.

What is the purpose of restriction analysis in the context of plasmid cloning?

To insert new genes into the bacterial genome.

To confirm the successful cloning of a new gene into the plasmid. (correct)

To induce mutations in the bacterial cells.

To measure the overall size of the plasmids without digestion.

How does the size of DNA fragments affect their migration speed during agarose gel electrophoresis?

Longer fragments migrate slower than shorter fragments. (C) Signup and view all the answers

Why is it important to select appropriate restriction enzymes when performing restriction analysis?

To ensure that the DNA combinations created will produce unique fragment patterns for different cloning results. (C) Signup and view all the answers

What is the primary function of the origin of replication (ORI) sequence in a plasmid?

To dictate the number of plasmid copies within a cell. (D) Signup and view all the answers

Which enzyme is responsible for cleaving the phosphodiester bond in a DNA strand?

Restriction endonuclease (A) Signup and view all the answers

What is the role of methyltransferase in the restriction-modification system?

To protect DNA through methylation. (A) Signup and view all the answers

What is a key characteristic of plasmids compared to bacterial chromosomes?

Plasmids are usually circular and very small. (C) Signup and view all the answers

What is the primary reason bacteria used for cloning have a modified restriction-modification system?

To eliminate the bacteria's restriction capabilities but maintain methylation activity. (C) Signup and view all the answers

Which feature of plasmids often provides an advantage to bacteria in a hostile environment?

The presence of antibiotic resistance genes. (B) Signup and view all the answers

In the context of the restriction-modification system, why is it important for a bacterium to methylate its own DNA?

To prevent its own DNA from being cleaved by restriction enzymes. (C) Signup and view all the answers

What is NOT a typical attribute of a plasmid mentioned in the text?

Contain genes vital for the bacteria's basic life-cycle. (D) Signup and view all the answers

Which type of restriction enzyme cleaves DNA at random locations far from its recognition sequence, often containing restriction-modification enzyme combinations?

Type I (C) Signup and view all the answers

Which of the following enzyme types is most frequently used in molecular cloning due to their ability to cleave DNA near or within palindromic recognition sequences?

Type II (B) Signup and view all the answers

A restriction enzyme is named EcoRI. What does the 'R' most likely stand for?

The bacteria strain (C) Signup and view all the answers

What is the average length of DNA fragments produced by a restriction enzyme that recognizes a six-nucleotide sequence specific for sequences containing GC in the human genome?

65000 bp (C) Signup and view all the answers

A Type III restriction enzyme requires what to recognize a sequence?

Non-palindromic sequences with opposite orientation (A) Signup and view all the answers

If a restriction enzyme recognizes a four-nucleotide sequence, what is the approximate average length of the DNA fragments it will generate?

260 bp (D) Signup and view all the answers

Why do fragments of various lengths result when DNA is cut with the same restriction enzyme?

Recognition sequences are not randomly distributed in the genome. (D) Signup and view all the answers

Type IIs restriction enzymes are known for which characteristic?

Cleaving at specific locations some distance from the recognition sequence, always on one side. (A) Signup and view all the answers

What is the primary function of T4 ligase in molecular cloning?

To catalyze the formation of a phosphodiester bond between DNA strands. (B) Signup and view all the answers

Which enzyme is used to prevent self-ligation of a plasmid during the cloning process?

Alkaline phosphatase (A) Signup and view all the answers

What is a distinguishing characteristic of S1 nuclease's activity?

It only digests single-stranded DNA and RNA. (C) Signup and view all the answers

Which enzyme has both polymerase and exonuclease activity and is used to create blunt DNA ends?

Klenow Fragment (A) Signup and view all the answers

What is the recognition sequence of the BamHI enzyme?

5’-GGATCC / 3’ CCTAGG (B) Signup and view all the answers

What is a key benefit of using artificial restriction enzymes compared to natural ones?

They recognize dramatically longer sequences of DNA. (A) Signup and view all the answers

Which end of a DNA strand is modified by alkaline phosphatase?

The 5’ end, removing the phosphate group. (A) Signup and view all the answers

The ligation process is most efficient when joining which types of DNA ends?

Sticky ends, which are stabilized by hydrogen bonds. (A) Signup and view all the answers

In the context of the cloning exercise, if the OVA-GFP insert has the same orientation as the CMV promoter, which clone would it most likely be?

Clone A (D) Signup and view all the answers

Considering the provided plasmid maps, if the OVA-GFP insert is oriented opposite to the CMV promoter, which clone is most likely represented?

Clone B (C) Signup and view all the answers

Which factor is least important when selecting restriction enzymes for restriction analysis?

The expense of the enzyme. (D) Signup and view all the answers

When calculating fragment masses after restriction enzyme digestion, how is the fragment length determined?

By finding the difference in distance (in base pairs) between the cleavage sites. (C) Signup and view all the answers

If a restriction enzyme cuts a circular plasmid at two sites, what is the minimum number of fragments produced?

2 (D) Signup and view all the answers

In the context of the plasmid maps, how many distinct bands would one expect to see on an agarose gel if the plasmid pUC OVA-GFP was digested with HindIII?

1 (C) Signup and view all the answers

According to the provided plasmid maps, which of the following is true of the 'Double symmetrically closed insert' plasmid?

It contains insert DNA in opposite orientations. (C) Signup and view all the answers

Why are closed DNA inserts in plasmids, as presented in the third set of plasmid maps, difficult to maintained in a host organism?

The lack of open DNA ends interferes with replication. (A) Signup and view all the answers

What is the primary purpose of conducting restriction digestion of plasmids in this study?

To analyze the size of DNA fragments (C) Signup and view all the answers

Which restriction enzyme among the following specifically targets the BamHI recognition sequence?

BamHI (D) Signup and view all the answers

If a host clone does not contain the insert, what might be a possible outcome during the electrophoresis of the plasmids?

A single band representing the vector (D) Signup and view all the answers

What does the presence of an insert in a plasmid typically indicate during a restriction analysis?

The unique fragment patterns compared to the vector (A) Signup and view all the answers

During the restriction analysis, what might the bands on the agarose gel represent?

DNA fragments resulting from enzymatic cuts (A) Signup and view all the answers

Study Notes

DNA Vectors (Plasmids)

Plasmids are extrachromosomal DNA elements that replicate independently in bacteria and some eukaryotes.
They are typically circular and double-stranded DNA.
Plasmid size ranges from 1 kb to over 100 kb.
The number of plasmid copies per cell is determined by the origin of replication (ORI) sequence.
Some plasmids can transfer horizontally between bacteria, often carrying antibiotic resistance genes.

Restriction Enzymes

Restriction enzymes are nucleases that cut DNA at specific sequences.
They are found in bacteria and some archaea.
They often work in combination with methylases that protect the bacteria's own DNA from being cut.
Different restriction enzymes recognize different palindromic sequences.
Type II restriction enzymes are commonly used in molecular cloning, as they cleave DNA at specific locations near or within their recognition sequences.
Restriction enzymes can produce "sticky ends" or "blunt ends"
Sticky ends have overhanging, single-stranded sequences.
Blunt ends have no overhanging sequences.
Methylation protects DNA from restriction enzymes.

Molecular Cloning

Molecular cloning is the process of isolating and amplifying specific DNA fragments.
The process involves:
- Preparing a DNA fragment, usually by restriction digestion.
- Preparing a plasmid vector.
- Ligation of DNA fragments into the vector.
- Transforming the ligated product into host cells (bacteria).
- Selecting host cells that have taken up the recombinant plasmid.
Commonly used tools in cloning :
- Restriction enzymes
- DNA ligase
- Alkaline phosphatase
- Klenow fragment

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Description

This quiz covers the essential concepts of DNA vectors, specifically plasmids, and the role of restriction enzymes in molecular biology. Learn about their structure, function, and significance in genetic engineering. Test your knowledge on how these tools are applied in cloning and gene manipulation.