Biotechnology Workshop Week 6

Questions and Answers

What is the primary function of a restriction enzyme?

To replicate DNA in a host cell

To cut DNA at specific sequences (correct)

To visualize DNA fragments

To synthesize DNA from RNA

What type of ends does a restriction enzyme generate when it cuts DNA?

Non-specific ends

Blunt ends or sticky ends (correct)

Double-stranded ends

Circular ends

What is the primary function of ethidium bromide (EtBr) in electrophoresis?

To create DNA fragments

To intercalate between DNA bases (correct)

To degrade DNA fragments

To enhance DNA replication

Which component of a cloning vector ensures that the vector can replicate independently within a host cell?

Origin of Replication Signup and view all the answers

Which method is used to visualize DNA fragments after gel electrophoresis?

Ethidium bromide Signup and view all the answers

What role does the antibiotic resistance gene (e.g., AmpR) play in plasmid selection?

It provides survival advantage on antibiotic media. Signup and view all the answers

During DNA gel electrophoresis, what determines the speed at which DNA fragments migrate?

The size of the DNA fragments Signup and view all the answers

What is a major difference between bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)?

BACs can carry up to 300,000 bp; YACs up to 1,000,000 bp. Signup and view all the answers

Which of the following describes a key regulatory element difference between expression vectors and cloning vectors?

Expression vectors have additional elements like ribosome binding sites. Signup and view all the answers

What is the purpose of the selectable marker in a cloning vector?

To identify cells that have taken up the vector Signup and view all the answers

What two characteristics of DNA fragments does gel electrophoresis utilize for separation?

Size and charge Signup and view all the answers

Which of the following is a consequence of using bacterial expression vectors for human gene proteins?

There is a risk of protein misfolding and aggregation. Signup and view all the answers

What process is used to introduce a recombinant plasmid into bacterial cells?

Transformation Signup and view all the answers

What is the main purpose of using expression vectors in genetic engineering?

To produce proteins encoded by the inserted genes. Signup and view all the answers

Which characteristic differentiates a cloning vector from an expression vector?

Cloning vectors replicate DNA; expression vectors produce proteins. Signup and view all the answers

How do the uses of YACs and BACs typically differ?

YACs are better for studying large genes and genomic regions. Signup and view all the answers

Study Notes

Restriction Enzymes

Proteins that cut DNA at specific sequences.
Make two cuts in double-stranded DNA (dsDNA) creating blunt ends (straight cuts) or sticky ends (staggered cuts with overhangs).
Useful in genetic engineering and molecular biology for fragment generation.

Cloning the Human Insulin Gene

Isolate the insulin gene by extracting mRNA from human pancreatic cells and converting it to complementary DNA (cDNA).
Prepare a vector by cutting a bacterial plasmid with a restriction enzyme to create sticky ends.
Ligate the insulin cDNA into the plasmid, creating a recombinant plasmid.
Transform bacterial cells with the recombinant plasmid and select for successful transformants.
Screen bacterial clones to confirm the presence of the insulin gene.
Induce growth and purify the produced insulin protein.

Characteristics of a Cloning Vector

Origin of Replication (ori): Enables independent replication within the host cell.
Selectable Marker: Typically an antibiotic resistance gene, allowing identification of cells that have taken up the vector.
Multiple Cloning Site (MCS): Contains unique restriction enzyme sites for easy insertion of foreign DNA.

DNA Gel Electrophoresis

Separation Characteristics: DNA fragments are separated based on size and charge.
Migration Process: DNA samples loaded in a gel move towards the positive electrode; smaller fragments migrate faster and further, forming distinct bands.
Visualization: Ethidium bromide (EtBr) is commonly used, intercalating with DNA and fluorescing under UV light for band visibility.

Selectable Markers in Plasmids

Antibiotic Resistance Gene: Provides survival advantage on antibiotic media, enabling selection of transformed bacteria.
LacZ Gene: Codes for β-galactosidase, turning blue in the presence of X-gal; white colonies indicate successful gene insertion.

Types of Vectors

Bacterial Artificial Chromosomes (BACs): Can carry large fragments (up to 300,000 base pairs); ideal for cloning extensive DNA segments in bacterial cells.
Yeast Artificial Chromosomes (YACs): Larger capacity (up to 1 million base pairs); useful for cloning very large DNA sequences in yeast cells.

Differences Between Expression and Cloning Vectors

Purpose: Cloning vectors amplify DNA; expression vectors produce proteins encoded by genes.
Regulatory Elements: Cloning vectors have basic elements; expression vectors include promoters and binding sites for protein synthesis.
End Product: Cloning vectors yield multiple DNA copies; expression vectors yield the protein product.

Drawbacks of Bacterial Expression Vectors for Human Genes

Lack of Post-Translational Modifications: Bacteria cannot perform complex modifications (e.g., glycosylation), essential for human protein function.
Protein Misfolding and Aggregation: Human proteins may misfold or aggregate in bacteria, resulting in non-functional proteins.

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Description

This quiz explores the concept of restriction enzymes and their role in genetic engineering. Participants will learn about how these enzymes function, the types of DNA fragments they generate, and their importance in molecular biology. Enhance your understanding of cloning techniques and DNA manipulation through this engaging quiz.