Molecular cloning

Question 2

Why do we want or need to clone genes?

Answer 2

• To study certain genetic diseases, e.g. cystic fibrosis
• To make it easier to study the proteins that the genes encode
• To enable scientists to manipulate and study genes in isolation from the organism they came from

Question 3

What does molecular cloning refer to?

Answer 3

The isolation of individual genes or other segments of DNA and moving them into an extrachromosomal DNA in another organism for replication.

Question 4

What are the main stages in molecular cloning?

Answer 4

1. Isolation and purification of the DNA or gene of interest
2. Insertion of the desired piece of DNA into a convenient carrier DNA molecule or cloning vector (vector, for short) for replication
3. Producing the vector in host organism(s)
4. Measurement of the efficacy of the cloning

Question 5

Define Chimera?

Answer 5

Any hybrid molecule of DNA that has been merged from two different sources of DNA, e.g. a vector plus a cloned gene.

Question 6

What is a cloing vector?

Answer 6

Any molecule of DNA that can replicate itself inside a cell and is used for carrying cloned genes or segments of DNA. They are usually a small multicopy plasmid or a modified virus.

Question 7

What is a plasmid?

Answer 7

An extrachromosomal circular piece of DNA that are found in various bacteria and even eukaryotes and is often used as cloning vectors for genetic engineering.

Question 8

What are the key properties of cloning vectors?

Answer 8

• Reasonably small and manageable DNA molecules
• Moving the vector from one host to another should be relatively easy
• Generating and purifying large amounts of vector DNA should be straightforward

Question 9

What are other factors to consider regarding cloning vectors?

Answer 9

• A mechanism to select host cells containing the vector
• Ability to insert genes into the vector
• Detect the presence of an inserted gene in the vector

Question 10

Plasmid feature: Antibiotic resistance gene

What is its purpose?

Answer 10

Resistant to the corresponding antibiotics: to separate the bacteria harbouring the plasmid from those without the plasmid during amplification.

Question 11

Plasmid feature: Selectable marker

What is its purpose?

Answer 11

Any gene that makes the host cell harbouring the plasmid have an easily identified trait to separate the harbouring cells from those without the plasmid, e.g. antibiotic resistance.

Question 12

Plasmid feature: Promoter region

What is its purpose?

Answer 12

• The promoter region is found upstream or before the site where the gene of interest is added.
• It directs RNA polymerase to make mRNA copies of the gene of interest

Question 13

Plasmid feature: Origin of replication (ORI)

What is its purpose?

Answer 13

The DNA sequence that ensures that the plasmid is replicated during the cell cycle and that each daughter cell gets one or more copies of the plasmid

Question 14

Plasmid feature: Multiple cloing sire (MCS) or polylinker insert

What is its purpose?

Answer 14

• A series of restriction enzyme sites that are used to connect the DNA fragment or gene of interest to the plasmid.
• The term used to describe the gene of interest or DNA fragment of interest that is joined to the plasmid

Question 15

Plamid feature: Primer binding site

What is its purpose?

Answer 15

• A DNA sequence that is complementary to the sequence of a polymerase Chain Reaction (PCR) primer for amplification of the insert.
• The primer binding site is also useful for traditional Sanger sequencing of the insert.

Question 16

Plasmid feature: Terminators

What are their purpose?

Answer 16

The terminator region defines the end of transcription, and ensures that RNA polymerase only transcribes the gene of interest.

Question 18

Label the key features of cloning vectors

Answer 18

Question 19

State the different types of cloning vectors

Answer 19

• Shuttle vectors a vector that can survive in more than one type host cells and facilitates cloned genes moving from on organism to another (probably most commonly used vectors)
• Transient Expression vectors- short term expression of gene of interest
• Stable Expression Vectors- allows stable expression of gene of interest throughout cell growth
• Lentiviral vectors- use viruses as an expression system
• Inducible vectors- You can induce the expression of the gene of interest (switch on and off)
• Bacteriophage lambda vectors
• Cosmid vectors
• Yeast artificial chromosomes (YACs)