Biotechnology Overview: Plasmids & Recombinant DNA

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Questions and Answers

What is the main purpose of recombinant DNA technology?

To create new plasmids without bacteria

To solely produce proteins

To enhance or suppress gene expression (correct)

To study bacterial growth patterns

Type II restriction endonucleases are not suitable for gene cloning.

False

What is a plasmid?

A circular self-replicating DNA molecule separate from bacterial DNA.

Restriction endonucleases are also known as __________.

restriction enzymes Signup and view all the answers

Match the following types of restriction endonucleases with their characteristics:

Type I = Cleaves DNA at sites other than the recognition site Type II = Widely used for gene cloning Type III = Also cleaves DNA at sites other than the recognition site Signup and view all the answers

Which of the following is NOT a use of recombinant DNA technology?

Synthesizing antibiotics Signup and view all the answers

What is one disadvantage of the single restriction method of inserting foreign DNA into plasmid vectors?

Difficult to achieve directional cloning Signup and view all the answers

The polylinker affects the transcription of the lac Z' gene.

False Signup and view all the answers

What two restriction enzymes are typically used in the double restriction approach mentioned?

BamHI and EcoRI Signup and view all the answers

The process of making the bacterial membrane transiently porous to allow plasmid entry is called __________.

transformation Signup and view all the answers

Match the following techniques with their descriptions:

Single restriction = Involves cutting with one enzyme allowing insert in both orientations Double restriction = Uses two enzymes to ensure correct insert orientation Transformation = Process of getting the recombinant plasmid into bacterial cells Directional cloning = Inserts DNA in a specific orientation relative to control sequences Signup and view all the answers

Which of the following is NOT a source of foreign DNA for cloning?

PCR-amplified RNA Signup and view all the answers

The plasmid pBR322 is an example of a high copy number plasmid.

False Signup and view all the answers

What does the lowercase 'p' in plasmid vectors stand for?

plasmid Signup and view all the answers

An ideal bacterial plasmid vector must contain an origin of _______ to allow independent replication.

replication Signup and view all the answers

Match the plasmid with its description:

pBR322 = Has antibiotic resistance genes pUC18 = High copy number plasmid pUC19 = Derivative of pBR322 Signup and view all the answers

What is a key feature of a multiple cloning site (polylinker) in a plasmid vector?

Contains unique restriction endonuclease cleavage sites. Signup and view all the answers

Stringent control of plasmid replication allows for many copies of the plasmid in each bacterial cell.

False Signup and view all the answers

What type of genetic selection is used to distinguish transformed host cells from untransformed ones?

Antibiotic resistance or blue-white screening Signup and view all the answers

Modern cloning vectors are often built from _______ plasmid vectors.

pUC19 Signup and view all the answers

What feature is crucial for the extraction of plasmids from host cells?

Efficient and simple extraction method Signup and view all the answers

What is the function of BamHI in recombinant DNA technology?

To cut DNA at specific sequences Signup and view all the answers

T4 DNA ligase is used to covalently link DNA fragments together.

True Signup and view all the answers

What are the complementary sticky ends generated by BamHI in the provided DNA sequences?

5’--G G-A-T-C-C--3’ and 3’--C-C-T-A-G G--5’ Signup and view all the answers

Terminal deoxynucleotidyl transferase adds deoxynucleotides to the ______ ends of DNA molecules.

3′OH Signup and view all the answers

Match the following enzymes with their functions:

BamHI = Cuts DNA at specific sequences T4 DNA ligase = Links DNA fragments together DNA polymerase III = Synthesizes DNA from a template Terminal transferase = Adds nucleotides to 3' OH ends of DNA Signup and view all the answers

What is a key characteristic of the DNA fragments generated by BamHI?

They have sticky or cohesive ends. Signup and view all the answers

DNA polymerase III and terminal transferase have the same requirements for DNA synthesis.

False Signup and view all the answers

What type of ends do the fragments have after cleavage with BamHI?

Sticky ends Signup and view all the answers

Study Notes

Plasmids

Circular DNA molecule, separate from bacterial DNA
Replicates independently
Can be used to introduce foreign DNA into bacteria

Recombinant DNA Technology

Used to study gene arrangement, expression and regulation
Modifies gene expression to enhance or suppress products
Creates artificial copies of DNA segments
Transfers genes between organisms, creating transgenic organisms with altered characteristics
Creates transgenic organisms used for commercial purposes

Restriction Endonucleases (REs)

Bacterial enzymes that cut DNA at specific recognition sites
Type II REs are used in gene cloning because they cleave DNA at specific sites
Important for mapping and reconstructing DNA in vitro

Terminal Deoxynucleotidyl Transferase (Terminal Transferase)

Enzyme that adds deoxynucleotides to the 3' end of DNA molecules
Does not have proofreading ability
Distinct from DNA polymerase III, which has proofreading ability

Foreign DNA Cloning Sources

Chromosomal DNA
mRNA converted to cDNA
PCR-amplified DNA

Cloning and Expression Vectors

Carry foreign DNA into bacterial cells
Used in gene cloning and protein production
Plasmids are named with a system of uppercase letters and numbers, "p" stands for plasmid
pBR322, pUC18, p UC19 are examples of commonly used plasmids
High copy number plasmids replicate more than 5oo copies per bacterial host cell, occur in plasmids with relaxed replication control
Stringent replication is coupled to host chromosome, results in lower copy numbers

Essential features of a bacterial plasmid vector

Origin of replication (ori) for independent replication
Selection marker/s for identifying transformed cells:
- Antibiotic resistance
- Blue-white screening
Multiple cloning site (MCS) containing restriction endonuclease cleavage sites for insertion of foreign DNA
Easy extraction from host cells

Inserting Foreign DNA into Plasmid Vectors

Single restriction approach:
- Can lead to ligation of the restricted plasmid
- Foreign DNA can be inserted in both orientations
- Multiple fragments can insert into the excised plasmid
Double restriction approach:
- Uses two restriction enzymes to cleave the plasmid and foreign DNA components
- Reduces problems associated with single restriction
- Directional cloning, ensures foreign DNA is inserted in the correct orientation with respect to transcriptional and translational control sequences

Bacterial Transformation

Process of introducing recombinant plasmids into bacterial cells
Makes bacterial membrane transiently porous for plasmid uptake

Cosmids:

Plasmids with a cos site (cohesive end site)
Can be packaged into phage heads due to the cos site
Can carry inserts of 33-47 kbp
Combine advantages of plasmids and phage vectors

Bacterial Artificial Chromosomes (BACs):

Based on the fertility plasmid (F plasmid)
Designed for cloning large DNA sequences
Contain partition genes for even distribution of plasmids during bacterial cell division
Accommodate inserts of up to 300 kbp
Used in chromosome mapping

Expression Vectors:

Designed for protein expression of target genes
Often plasmid-based
Utilize T7 RNA polymerase gene expression system for efficient production of proteins
Contain transcriptional and translational control sequences for gene expression
Transform E.coli host cells with a T7 RNA polymerase gene integrated into its chromosome

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Description

Explore the fundamental concepts of plasmids, recombinant DNA technology, and the role of restriction endonucleases. This quiz covers how these elements work together to modify gene expression and create transgenic organisms for various applications. Test your understanding of key enzymes and their functions in molecular biology.

Biotechnology Overview: Plasmids & Recombinant DNA

Podcast Beta

Questions and Answers

What is the main purpose of recombinant DNA technology?

Type II restriction endonucleases are not suitable for gene cloning.

What is a plasmid?

Restriction endonucleases are also known as __________.

Match the following types of restriction endonucleases with their characteristics:

Which of the following is NOT a use of recombinant DNA technology?

What is one disadvantage of the single restriction method of inserting foreign DNA into plasmid vectors?

The polylinker affects the transcription of the lac Z' gene.

What two restriction enzymes are typically used in the double restriction approach mentioned?

The process of making the bacterial membrane transiently porous to allow plasmid entry is called __________.

Match the following techniques with their descriptions:

Which of the following is NOT a source of foreign DNA for cloning?

The plasmid pBR322 is an example of a high copy number plasmid.

What does the lowercase 'p' in plasmid vectors stand for?

An ideal bacterial plasmid vector must contain an origin of _______ to allow independent replication.

Match the plasmid with its description:

What is a key feature of a multiple cloning site (polylinker) in a plasmid vector?

Stringent control of plasmid replication allows for many copies of the plasmid in each bacterial cell.

What type of genetic selection is used to distinguish transformed host cells from untransformed ones?

Modern cloning vectors are often built from _______ plasmid vectors.

What feature is crucial for the extraction of plasmids from host cells?

What is the function of BamHI in recombinant DNA technology?

T4 DNA ligase is used to covalently link DNA fragments together.

What are the complementary sticky ends generated by BamHI in the provided DNA sequences?

Terminal deoxynucleotidyl transferase adds deoxynucleotides to the ______ ends of DNA molecules.

Match the following enzymes with their functions:

What is a key characteristic of the DNA fragments generated by BamHI?

DNA polymerase III and terminal transferase have the same requirements for DNA synthesis.

What type of ends do the fragments have after cleavage with BamHI?

Study Notes

Plasmids

Recombinant DNA Technology

Restriction Endonucleases (REs)

Terminal Deoxynucleotidyl Transferase (Terminal Transferase)

Foreign DNA Cloning Sources

Cloning and Expression Vectors

Essential features of a bacterial plasmid vector

Inserting Foreign DNA into Plasmid Vectors

Bacterial Transformation

Cosmids:

Bacterial Artificial Chromosomes (BACs):

Expression Vectors:

Studying That Suits You

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