Genetic engineering

Question 1

What are the ingredients of PCR reaction

Answer 1

Template, primers, dNTPS, buffer (Mg2+), Taq polymerase

Question 2

What are the 3 steps in PCR>

Answer 2

Denaturation, Annealing - primers bind to DNA and polymerase attaches, Extension

Question 3

What type of application is PCR?

Answer 3

exponential

Question 4

What section is amplified?

Answer 4

DNA between primers - ends of the amplified fragment are defined by 2 primers

Question 5

Describe PCR primers

Answer 5

short 20bp single stranded DNA chemically synthesised

Question 6

Describe amplification

Answer 6

30 cycles of PCR - 10^6 fold

1pg -> 1yg DNA enough to analyse on gel

Question 7

Analysis of DNA - agarose gel electrophoresis

Answer 7

Fractionation of DNA by size
DNA moves to positive electrode through gel depending on shape. and size
Stain DNA with dye for UV exposure

Question 8

Analysis of DNA - sequencing

Answer 8

PCR products can be directly sequenced

Question 9

What are the users of PCR?

Answer 9

Any research uses in which a specific piece of DNA needs to be amplified
• DNA sequencing
• Detection of pathogens in water, blood
• Genetic fingerprinting
• Forensic analysis
• Diagnosis of genetic disorders
• Prenatal diagnosis
• Analysis of ancient DNA

Question 10

What are the limitations of PCR

Answer 10

Sequenceinformationisrequiredto design 2 primers
2. Limitonlengthofamplifiedfragment
3. Significantinvitromutationrate:
Taq pol. ~10-4
4. Verysensitivetoexactreaction
conditions, so not easily quantified
5. Tinyamountsofcontaminating DNA will also be amplified

Question 11

How can DNA be expressed in cells?

Answer 11

1. IntroductionofDNAintocell
2. Replicationandexpressionin the cell
   Can express a gene in bacteria by inserting it into a plasmid.

Question 12

Describe recombinant DNA

Answer 12

Circular double-stranded plasmid DNA cloning vector – cleavage with restriction nuclease, DNA fragment to be cloned – covalent linkage by DNA ligase

Question 13

Describe cutting DNA

Answer 13

DNA is cut by restriction endonuclease - enzymes extracted from bacteria
Recognised and cuts at the sequence

Question 14

Describe joining DNA

Answer 14

DNA ligase, forms phosphdiester bonds, requires ATP,any two DNA molecules can be joined

Question 15

Gene cloning

Answer 15

Extract DNA from organism, cut DNA, open up vector, make recombinant DNA, introduce into bacteria, clones

Question 16

Gene library

Answer 16

Collection of recombinant clones
Can screen for clones containing gene of interest
Various methods e.g. techniques involving DNA/RNA hybridisation

Question 17

How is cloning of very large DNA inserts possible?

Answer 17

Yeast Artificial Chromosomes

Question 18

Transgenics

Answer 18

Genes between species, genetic code is universal, expression is possible, for eukaryotic genes normally need to make cDNA copy of mRNA

Question 19

Name some useful products produced using genetic engineering

Answer 19

Human insulin
Blood clotting factor VIII
Human growth hormone
Bovine chymosin (for vegetarian cheese) Hepatitis B vaccine
Artemisinin (anti-malarial)