Recombinant DNA Technology

What is recombinant DNA technology?

A set of techniques used to manipulate DNA

What is the purpose of recombinant DNA technology?

To create new DNA combinations for gene cloning and protein production

What is reverse transcriptase?

An enzyme that synthesises DNA from an RNA template

Where is reverse transcriptase found naturally?

In some viruses and bacteria

Why is reverse transcriptase useful in genetic engineering?

It allows DNA copies of genes to be made from mRNA

Why is mRNA used to make DNA copies of genes?

It contains only coding regions with no introns

What is complementary DNA (cDNA)?

DNA made from an mRNA template using reverse transcriptase

Why is cDNA useful?

It acts as a functional gene that can be inserted into vectors

What are restriction endonucleases?

Enzymes that cut DNA at specific base sequences

Where do restriction endonucleases come from?

Bacteria

What is a recognition site?

A specific DNA sequence where a restriction enzyme cuts

How long are most recognition sites?

About six base pairs

What are sticky ends?

Overhanging ends of DNA formed by staggered cuts

What are blunt ends?

Straight cuts that leave no overhangs

Why are sticky ends useful?

They allow complementary base pairing between DNA fragments

How are complementary sticky ends produced?

By cutting DNA with the same restriction endonuclease

Why do complementary sticky ends join easily?

They base-pair before covalent bonds form

What enzyme permanently joins DNA fragments?

DNA ligase

What bonds does DNA ligase form?

Phosphodiester bonds

What is a vector?

A carrier used to transfer DNA into a cell

Why are vectors needed?

Naked DNA would be digested by enzymes inside cells

What are plasmids?

Small circular DNA molecules found in bacteria

Why are plasmids used as vectors?

They naturally replicate in bacteria

What is in-vivo gene cloning?

Inserting DNA into living cells to make many copies

What is the first step of inserting a gene into a plasmid?

Cutting both gene and plasmid with the same restriction enzyme

Why must the same restriction enzyme be used?

To produce complementary sticky ends

What happens when the gene and plasmid are mixed?

Sticky ends base pair

What seals the gene into the plasmid?

DNA ligase

What is recombinant DNA?

A plasmid containing foreign DNA

What is a recombinant plasmid?

A plasmid with a new gene inserted

What is electroporation?

A method used to get plasmids into bacterial cells

How does electroporation work?

It increases membrane permeability

How is electroporation achieved?

Using calcium salts and rapid temperature change

What are transgenic microorganisms?

Microorganisms containing foreign DNA

What are gene markers?

Genes used to identify transformed cells

Why are gene markers used?

To identify bacteria that have taken up plasmids

What types of gene markers exist?

Antibiotic resistance, fluorescent, and enzyme markers

How do antibiotic resistance markers work?

Only bacteria with the plasmid survive on antibiotic media

How can gene markers show if the desired gene is inserted?

The marker gene becomes inactivated

What is an example of an antibiotic marker?

Ampicillin or tetracycline resistance