DNA Technology

Question 1

How do you isolate genes

Answer 1

Use enzymes
Either
Reverse transcriptase 
Or
Restriction endonuclease

Question 2

How is reverse transcriptase used to isolate genes

Answer 2

Locate and extract gene
mRNA carries the code
mRNA acts as template for cDNA
cDNA formed using RT enzyme
DNA polymerase binds complementary nucleotides on cDNA template
Double stranded DNA formed using cDNA template
Copy of desired gene in DNA

Question 3

How is restriction endonuclease used to isolate genes

Answer 3

Cuts DNA at recognition site
Cut leaves sticky ends so bigger bonding site
Complementary bases pair up
Enzyme DNA ligase joins them

Question 4

What is In Vivo gene cloning

Answer 4

Transferring fragments to a host cell using a vector

Question 5

What ways can DNA be inserted

Answer 5

Using plasmid vector
Using virus
Coat in lipids

Question 6

What is the role of a vector during in Vivo gene cloning

Answer 6

Transfers genes from one organism into another

Question 7

What vector is used to insert DNA into host cell

Answer 7

Plasmid

Question 8

What is transformation

Answer 8

Reintroducing the plasmid back into bacterial cells

Question 9

Describe the process of transformation

Answer 9

Mix together in medium containing calcium ions
Calcium ions and temperature change
Causes bacterial cells to become permeable
Plasmid passes through membrane into cytoplasm

Question 10

Why do not all bacteria cells have the DNA fragment

Answer 10

Only a few take up plasmids when mixed together

Some plasmids close up before DNA fragment is incorporated

Question 11

How do you identify which host cells have taken up the gene

Answer 11

Enzyme gene marker
Fluorescent gene marker
Replica plating using antibiotic resistance markers

Question 12

What happens when doing antibiotic resistance markers

Answer 12

Use plasmid with 2 antibiotic resistant genes
Insert desired gene into bacteria
Do replicate plate
Put one plate with disease
If survive, shows resistant to gene which shows did not take up wanted gene
Ones we want die so know which ones on non-replica plate to keep and grow

Question 13

How are fluorescent gene markers used

Answer 13

Jellyfish have green fluorescent protein

Transfer desired gene into plasmid from jellyfish

If bacteria cell taken up gene then they won’t fluoresce when viewed under microscope

Question 14

How are enzyme gene markers used

Answer 14

Gene marker codes for enzyme eg lactase
Lactase turns substance from clear to blue
Desired gene placed in centre of lactase gene
If gene present, won’t cause colour to change to blue in presence of lactose

Question 15

Why are fluorescence and enzyme gene markers more efficient than antibiotic ones

Answer 15

Won’t kill ones we want

So it’s quicker method

Question 16

When growing bacteria, what needs to be controlled

Answer 16

Temperature

PH

Question 17

What is In Vitro gene cloning

Answer 17

Polymerase chain reaction

Question 18

What is a thermocycler

Answer 18

Controls temperature changes

Question 19

What is polymerase chain reaction used for

Answer 19

Crime scene investigators

Info gaining about long dead organisms

Determine to identify father

Question 20

What are primers used for

Answer 20

Keep strands apart

Question 21

What is DNA polymerase used for in polymerase chain reaction

Answer 21

Join together nucleotides

Question 22

In Vitro

How are strands broke apart

Answer 22

Temperature

Break hydrogen bonds

Question 23

In Vitro

Why is heated to 95 degrees

Answer 23

To spear ate DNA strands

Question 24

In Vitro

Why is cooled to 55 degrees

Answer 24

Allowed primers to anneal

And primers to attach to start and end complementary bases on DNA strands

Question 25

In Vitro | Why is heated to 72 degrees

Answer 25

Optimum temperature for DNA polymerase

Question 26

Describe polymerase chain reaction process

Answer 26

``` Put in thermocycler Heated to 90 degrees to break bonds Cooled to 55 degrees So primers can be used Heated to 72 degrees Nucleotides bind to complementary bases DNA polymerase ``` Now have 2 DNA molecules Cycle takes 2 minutes Cycle keeps going

Question 27

What are the 5 main stages of DNA technology

Answer 27

``` Isolation Insertion Transformation Identification Growth/cloning ```

Question 28

Advantages of In Vivo

Answer 28

No risk of contamination Very accurate Makes copies of specific gene not whole DNA sample

Question 29

Disadvantage of In Vivo

Answer 29

Takes longer than In Vitro to produce same quantity

Question 30

Advantages of In Vitro

Answer 30

Doesn't require living cells | Very quick process

Question 31

Disadvantage of In Vitro

Answer 31

Requires very pure sample as could contaminate with other DNA, leading to a false result

Question 32

Benefits of genetic modification

Answer 32

``` Increase yield from crop Improve nutrient content in food Introduce resistance to disease Making vaccines Produce medicine for treating diseases Develop tolerance to environmental conditions ```

Question 33

Describe process of Anti-thrombin production

Answer 33

Egg and sperm Put gene in fertilised egg before division occurs Offspring with gene are crossbred Extracted and purified

Question 34

Benefits of recombinant DNA technology

Answer 34

Genetic fingerprinting used in forensic science Gene therapy to cure genetic disorders Genetically modified crop has economic and environmental advantages

Question 35

Risks of recombinant DNA technology

Answer 35

Don't know long term effects Gene may pass to different organisms, causes problems What is genetically engineered gene mutates? Expensive. Money better use eg fighting world hunger

Question 36

how can cystic fibrosis be treated using gene therapy

Answer 36

gene replacement gene supplementation germ-line gene therapy somatic-cell gene therapy

Question 37

what is germ-line gene therapy

Answer 37

replacing defective gene in fertilised egg so cells of offspring will develop normally

Question 38

what 2 methods are used to deliver genes in gene therapy

Answer 38

``` harmless virus (adenovirsues) wrapping it in lipids ``` both then inserted in patient's nostrils

Question 39

why are forms of delivering gene therapy not always effective

Answer 39

adenoviruses may cause infections patient may develop immunity to adenoviruses even if delivered successfully, few genes actually expressed

Question 40

How would you use gene therapy to treat severe combined immunodeficiency

Answer 40

Normal enzyme isolated from healthy human tissue Then inserted into retrovirus Retrovirus mixed with patient's T cells Retrovirus inject copy of normal enzyme gene into T cells T cells reintroduced into patient's blood, now with genetic code needed to make enzyme

Question 41

What is somatic-cell gene therapy

Answer 41

Gene therapy that targets just the affected tissue Healthy genes are not passed on to future generations Means treatment needs to be repeated