Heaphy 11 Genetic engineering

Question 1

Recombinant DNA

Answer 1

(genetic engineering) extends traditional biotechnology.

• ability to cut & join heterologous DNA. Relies on restriction enzymes.

Question 2

Recombinant DNA Used for:

Answer 2

• Botox, protein from C. botulinum -> lethal to paralyse, can be clinical use to stop muscle contractions.
• Can be purified from anerobic cultures Or from engineered E.coli
• Often carried out in micro-organisms. Bacteria E. coli, yeast.
• Cheap well understood. Both have autonomously replicating plasmids as cloning vectors

Question 3

Requires:

Answer 3

RESTRICTION ENZYMES

• Cloning vectors
• Other enzymes used in cloning
• PCR
• Protein production

Question 4

RECOMBINANT DNA CORE TECHNIQUE

Answer 4

Sticky ends cut, then stuck together

Compatible cohesive ends

T4 DNA Ligase enzyme +ATP ligate the ends together

• Plasmid can be amplified to prepare enormous amounts of cloned DNA if desired

Question 5

EcoR1:

Answer 5

restrict, sticky ends, compatible, base pair with each other, anneal, covalently linked => cloned, amplified

Can self ligate to create mismatch => titrations to ensure the major product is desired plasmid w/ insert

Question 6

Vectors

Answer 6

amplify DNA
Have marker gene => resistance to antibiotics
Grow on plates with antibiotics

Question 7

Enzymes of genetic engineering

ENDOnucleases:

Answer 7

• Most important restriction enzymes- cut DNA molecules at internal sites).
• Restriction enzymes isolated and characterised from hundreds of bacterial species have different specificities i.e. recognition sequences for cutting:

Question 8

• HindIII

Answer 8

from Haemophilus influenzae A*AGCTT blunt ends

Question 9

• EcoRI

Answer 9

from Escherichia coli G*AATTC compatible cohesives

Question 10

BamHI

Answer 10

from Bacillus amyloliquefaciens G*GATCC large fragments

Question 11

• Sau3A

Answer 11

from Staphylococcus aureus *GATC small fragments

Question 12

• Sma I

Answer 12

from Serratia marcescens CCC*GGG make blunt ends

Question 13

• All palendromes *

Answer 13

shows cutting point.

•

Question 14

DNA Ligase

Answer 14

rejoins the cut DNA

Phage T4. Requires ATP.

Question 15

Why genetically engineer?

Answer 15

• ‘engineered’ to contain recombinant DNA molecules comprising genes. Usually to express ‘foreign’ proteins, but sometimes RNA.
• Mutations could be ‘corrected’

Question 16

Uses of genetic engineering:

x7

Answer 16

• Vaccines: surface protein of pathogen used to immunise without symptoms or side-effects.
• human insulin humulin: for diabetics, previously animal (associated allergy problems
• human growth hormone: protropin, humatrope, dwarfism
• interferons: a therapeutic. -> MS, Hep C
• Factor VIII: for haemophiliacs
• Iinsecticidal toxin of Bacillus thuringiensis; kill insect pests but cannot spread to natural insect population.
• botox

Question 17

CLONING VECTORS

ORIGINS

Answer 17

• Developed from research on genetics, biochemistry and physiology of bacteria and bacterial viruses (phages) i.e. microbiology
pUC 19
- derived from plasmids or phage

Question 18

CLONING VECTORS

Function:

Answer 18

• replicate stably & independently of the host DNA
• easily separated from host DNA
• easily introduced into a host by transformation or infection
• may carry selectable markers , antibiotic resistance gene
• restriction enzyme cutting sites for cloning
• efficient promoters for expression of cloned genes.

Question 19

Phage vectors

Answer 19

• λ (lambda).
• M13
• Different vectors used for different purposes.
• very popular for making DNA libraries.
• M13 chromosome is single stranded DNA, used directly for DNA sequencing by Sanger method.

Question 20

DNA polymerase

Answer 20

(usually E. coli DNA polymerase I) Thermostable DNA polymerase (e.g. Taq from Thermus aquaticus) active at high temperatures, is used in polymerase chain reaction (PCR).

Question 21

Modifying enzymes

Answer 21

• phosphorylases, phosphatases other nucleases, etc. 5’ – OH grp +/ -
• reverse transcriptase (from retroviruses) copies RNA template into cDNA. So labile mRNA converted to stable DNA.

Question 22

PCR

Process:

Answer 22

•	DS DNA
•	Add primers
•	Anneal at 50’C
•	Polymerase copy DNA 72’C
•	Raise temp 
•	Repeat = clonal amplification
Can introduce unique restriction sites

Question 23

PCR
Uses:
x4

Answer 23

• Forensics
• Diagnostics
• DNA manipulation for cloning
• RT-PCR. Capture mRNA

Question 24

Protein production
uses
x3

Answer 24

• Design gene for human growth hormone.
• PCR from existing DNA
• Chemical synthesis from scratch? Codon usage.

Question 25

Protein production

Process

Answer 25

• Extract protein from cells, analyse, identify, purify

Question 26

PRODUCTION VECTORS

Answer 26

Not a single best choice. -> dependant on use e.g. virus for gene therapy

Question 27

Xenotransplantation

Answer 27

introduction of foreign viruses

Question 28

Gene therapy uses…

Answer 28

viral vectors

Question 29

Disadvantages of genetic engineering

Answer 29

Production problems- bacteria, insects, post translational differences.
Safety problems, chemical contamination, biological contamination