Gene Technology GMO

Question 1

GMO

Answer 1

Genetically Modified Organism

Question 2

Gene Transfer

Answer 2

Transfer of gene from a donor org to a recipient org

Involves combining DNA from different sources - recombinant DNA technology

Question 3

GEM

Answer 3

Genetically Engineered Microorganisms

Question 4

2 methods of obtaining required gene for GEM

Answer 4

•using restriction endonuclease to cut gene out of chromosomal DNA

•using reverse transcriptase to produce DNA from mRNA

Question 5

Using restriction endonuclease to cut gene out of chromosomal DNA

Answer 5

•chrom DNA cut into fragments using appropriate restriction endonuclease enzyme - cuts either side gene

•DNA fragment containing req gene is identified using comp gene probe

•Restriction endonuclease that cuts in staggered fashion prod sticky ends - most useful

If vector opened w/same restriction enzyme-exposed bases both comp - more readily base pair

Question 6

Using reverse transcriptase to produce DNA from mRNA

Answer 6

•mRNA obtained from cells where gene concerned is actively synthesising protein

•reverse transcriptase uses mRNA as template to produce comp single strand of DNA from free nucleotides

•Double stranded DNA made from cDNA using enzyme DNA polymerase - only double stranded can be annealed to the d-s plasmid vector

Question 7

Vector

Answer 7

Delivery tool to carry the gene into the host cell where it can be replicated and expressed

Question 8

Main types of Vector

Answer 8

Bacterial Plasmids
Viruses

Question 9

Bacterial Plasmids as Vector

Answer 9

genes into bacterial cells

•plasmid cut open using same restriction enzyme used to cut the DNA fragment - sticky ends of 2 types DNA contain comp base sequences (if mRNA synth ends are added)

•plasmid DNA and gene DNA anneal - H bonds form readily btwn sticky ends and DNA Ligase catalyses formation of phosphodiester bonds between sugar-phosphate backbones of both DNA - gene spliced into plasmid

Question 10

Recombinant DNA/Plasmid

Answer 10

DNA/plasmid that has foreign DNA inserted into it

Question 11

Viruses as Vectors

Answer 11

Adapted to insert genetic material into host cell

e.g. bacteriophage injects DNA into a bacterium - bacteriophage w/ DNA fragment spliced will transfer that recombinant DNA into bacterial cell

Question 12

Plasmid

Answer 12

Small circular piece of extrachromosomal DNA that occurs in certain bacteria

Carry extra genes

Question 13

Inserting vector into a host cell

Answer 13

Bacterial cells naturally take up plasmids - allowing exchange of genes

More readily when induced - incubated with Calcium Ions and treated with heat shock (0-42) - CSM more permeable to plasmids

Short electric pulse may be used to open pores in CSM (electroporation)

Bacteriophages effective way of delivering large genes to bacterial cells

Question 14

Transformed Bacteria

Answer 14

Bacteria that have taken up recombinant plasmid

Question 15

2 Ways of identigying successful take-up

Answer 15

Using Antibiotic-Resistant Marker Genes

Using Fluorescent Marker Genes

Question 16

Using Antibiotic-resistant Marker for Identification

Answer 16

R-plasmids carry genes that confer antibiotic resistance to bacteria

Restriction enzyme cuts in middle tetracycline resistance gene and desired inserted - active ampicillin resistance gene but inactive tetracycline resistance

-failed take up sensitive to both a and t
-take up of original plasmids resistant to both
-take up of recombinant resistant to ampicillin but not T

Question 17

Expirement AB-Resistant marker ID

Answer 17

Bacteria cultured on agar plates
-each bacterium multiplies to form colony

Colonies then replica-plated onto plate containing Ampicillin and a plate containing Tetracycline

Blotting og plate with a pad and pressing this against surface new plate

Bacteria that survive on Ampicillin are only transformed bacteria

Question 18

Using Fluorescent Marker Genes as Identification

Answer 18

More recent

Some org posses genes that produce fluorescent proteins - gene isolated and attached to gene of interest

Attached genes inserted into vector and carried into b cell
- take up readily identified as Transformed Bacteria will glow in UV light

Transformed then transferred to sterile nutrient medium and allowed to increase in number - gene cloning

Question 19

Genetically Modified Plants, how foreign genes inserted

Answer 19

•Agrobacterium Tumefaciens, invades damaged plant tissue and causes tumour-like growths - tumour inducing (Ti) plasmid splices desired gene - take up provided cellulose wall removed by cellulase enzyme

•Gene Gun - minute pellets covered in FNA shot through cellulose walls

•Plant Viruses - infect cells by inserting nucleic acid - new genes can be transferred

Question 20

Genetically Modified Animals Introduction of Genes

Answer 20

•Electroporation - CM temp disrupted by high voltage shock

•Liposomes - coat DNA which may adhere to CSM and pass DNA similar to Endocytosis

•Microinjection - inject DNA directly into nucleus of a fertilised egg

Question 21

Types of Viruses Used to Introduce Genes in GM Animals

Answer 21

Adeno - resp diseases, altered so that host cells not destroyed-useful for delivering genes to patients gene therapy

Retro - RNA delivered into host cell - copied to DNA and DNA incorporated into hosts chromosome

Question 22

Model Organsism

Answer 22

Species extensively studied to understand particular processes with the expectation that discoveries provide insight into workings over other organisms