PCR and Gene Cloning

Question 1

Explain what Polymerase Chain Reaction is

Answer 1

In vitro amplification is used to make lots of copies of a specific piece of DNA in a test tube

Question 2

Explain some of the uses of amplifying DNA

Answer 2

Sequencing genomes

Identifying infective agent

Detect inherited disorders

Detect genetic risk factors

Question 3

Name the 3 stages of DNA Amplification using PCR

Answer 3

DNA denaturation at 95 degrees C

Hybridization of primers at 50 - 65 degrees C

DNA elongation at 72 degrees C

Question 4

What are the resources needed for PCR?

Answer 4

DNA template

Primers - designed to amplify a specific sequence

DNA polymerase - heat resistant Taq polymerase often used

Nucleotides

Question 5

Explain briefly how agarose gel electrophoresis works

Answer 5

Place gel in buffer

Attach electrodes

Load samples

Apply current

Question 6

Name the 3 variations of PCR

Answer 6

Reverse Transcriptase (RT)-PCR - Analysing the expressed genome

PCR-Restriction fragment length polymorphism (RFLP)

DNA microarrays - Personal genomes

Question 7

Explain what the use of Recombinant DNA technology

Answer 7

Technology that allows DNA to be produced by artificial means

The introduction of manipulated genetic material into a cell in such a way as to allow it to replicate and be passed to progeny cells

Question 8

Explain the use of Gene Cloning

Answer 8

Generate multiple copies of a gene (DNA sequence) of interest

Move genes to alternate hosts

Analyze the gene in an easy-to-grow/easy to study host

Typical hosts: Escherichia coli or yeast cells

Question 9

Give an example of some synthetic protein products and their uses

Answer 9

Human Insulin - Treatment of diabetes

Human Growth hormone - Treatment for growth defects

Epidermal growth factor - Treatment for burns, ulcers

Tumor necrosis factor - Killing of some tumors

Interleukin-2 - Possible treatment for cancer

Question 10

Give the 3 main steps involved in Gene Cloning

Answer 10

Cut out DNA of interest

Insert DNA into a vector

Insert vector into a host

Question 11

Name the 3 enzymes that can be used to cut DNA and briefly explain them

Answer 11

Endonucleases - enzymes that cut inside a DNA molecule

Restriction endonucleases - recognize specific DNA sequences then cut at or near the sequence

Type II restriction enzymes - recognize and cut at highly specific targets

Mainly isolated from bacteria

Question 12

Name the sequence Restriction Endonucleases that recognize and how they cur the sequence

Answer 12

Palindrome ( GAATTC AND CTTAAG) and (GGCC AND CCGG)

Cut can be cut symmetrically or asymmetrical

Question 13

Name the 3 types of Vectors and their uses

Answer 13

Plasmids

Bacteriophages

Artificial chromosomes

Used for cloning large fragments, e.g. for preparing physical maps of genomes

Question 14

Explain the role of plasmid vectors

Answer 14

Extrachromosomal pieces of DNA capable of replication

Often circular

Found in bacteria and yeast

Often carry antibiotic resistance genes

Small plasmids often present in multiple copies

Genes cloned into plasmid will be present in multiple copies

Question 15

Explain what a pUC vector is

Answer 15

General purpose vectors

Multiple cloning site (polylinker)

Directional cloning

Colour screening

Question 16

Explain what a bacteriophage vector is

Answer 16

Bacterial viruses

Consists of a protein coat surrounding a nucleic acid

DNA containing bacteriophage used for cloning

Cloned DNA inserted into the phage genome

On infection of the host - multiple copies of the gene made

Question 17

Explain the process of a vector being inserted into a host and how this creates many copies

Answer 17

Cut and ligate DNA in a test tube

Insert plasmid into bacteria = transformation

Grow bacteria to produce millions of bacteria and multiple copies of the insert