Gene technologies

Question 1

What is the purpose of PCR

Answer 1

(Polymerase chain reaction)
to amplify quantity of DNA

Question 2

what are the 4 items required for PCR

Answer 2

-DNA sample to be amplified
-excess of 4 nucleotide bases (to synthesise new DNA strand)
-Tag DNA polymerase- bind nucleotides together
-Primers- provide starting sequence for Tag

Question 3

Stage 1 PCR

Answer 3

Separating DNA Strands
Temp: 90, 15s
Unzips polynucleotides by breaking H bonds between NCB

Question 4

What is the name and function of computer used in PCR

Answer 4

Thermal cycler
-Varies temp at precise time intervals

Question 5

Stage 2 PCR

Answer 5

Anneling Primers
Cooled to 55 degrees, 25s
Primers bond to 3’ end of DNA strands using complementary base pairing

Question 6

Stage 3 PCR

Answer 6

Synthesis
Heated to 75 (optimum temp for Taq), 80s
-Adds nitrogen containing bases to primers, building up complementary DNA strands
-Phosphodiester bonds form between adjacent nucleotides

-> Double stranded DNA identical to the original sequence

Question 7

Uses of PCR

Answer 7

-Forensic science
-DNA profiling in paternity testing
-DNA amplification of DNA extracted from fossils

Question 8

Similarities and differences between PCR and SCR

Answer 8

+free nucleotides
+ one new, one old strand in each molecule of DNA

-PCR produces only short strands (whole chro in SCR)
-PCR requires primers
-PCR requires high temps to separate DNA strands (DNA helicase in SCR)

Question 9

Intron

Answer 9

non-coding DNA

Question 10

exon

Answer 10

coding DNA- for ppc

Question 11

What are variable number tandem repeats

Answer 11

Patterns of repeated adjacent nucleotides

Question 12

why does Tag DNA polymerase not denature at 90 degrees

Answer 12

it has covalent bonds and disulphide bridges

Question 13

Use of VNTRs

Answer 13

• genetic fingerprinting
  -inheritance matching (more distant, less relatedness)
  -Essential for forensic science

Question 14

what is the function of electrophoresis

Answer 14

preparation and analysis of DNA for sequencing

Question 15

How to DNA fragments separate during electrophoresis

Answer 15

-DNA fragments put into wells in aragose gel
-Buffer solution added
-Current applied
-Since DNA fragments are - (phosphate groups), they move to positive end
-Shorter fragments of DNA move faster and further

Question 16

How can DNA bands be visualised following electrophoresis

Answer 16

Using fluorescent dye and a UV light

Question 17

Examples of GE in humans

Answer 17

Production of human insulin
Human growth hormones to treat dwarfism

Question 18

What is the def of GE

Answer 18

the manipulation of an organism’s DNA

Question 19

1 stage of GE: Gene Isolation

Answer 19

-Gene is isolated using restriction enzymes
-mRNA extracted from cells
-Reverse transcriptase produces single stranded complementary DNA (cDNA)
-DNA polymerase then produces strand complementary to cDNA
-end up with double stranded DNA

+double stranded DNA has no introns as produced from mature mRNA

Question 20

2 stage of GE: Insertion of gene into vector

Answer 20

-Vector eg. plasmid cut with same restriction enzymes
-Both plasmid and recombinant gene has complementary sticky ends
- They are annealed
-DNA ligase forms phosphodiester bonds between nucleotides of plasmid and recombinant gene-> recombinant plasmid

Question 21

3 stage of GE: Uptake of recombinant gene

Answer 21

-Put in Ca2+ solution and heat
-CSM becomes more permeable so recombinant plasmid taken up by bacteria
-Forms transgenic bacteria

Question 22

Identifying transgenic bacteria

Answer 22

-Reporter gene is also inserted into bacterium
-Recombinant gene is usually inserted into reporter gene so that it does not work
-cells not displaying reporter gene property have taken up gene

Question 23

Restriction enzymes

Answer 23

-Cuts DNA strand at specific site
-site complementary to active site of enzyme
-produce sticky ends

Question 24

What is meant by palindromic

Answer 24

sequence of bases on one strand is reversed on the other strand

Question 25

GE in cows

Answer 25

Lysozyme- antibacterial properties

Question 26

GE in goats

Answer 26

antithrombin- prevents blood clotting

Question 27

Advantages of using transgenic plants to produce human proteins

Answer 27

-low production costs -unskilled labour sufficient to maintain crops -minimal risks of contamination of human pathogens

Question 28

Crops have been genetically modified to:

Answer 28

-resistance to herbicides -produce insecticides -produce vitamin A

Question 29

How genes are inserted into GMC

Answer 29

- T-DNA, Ti plasmid inserted into bacterium -Gene gun- DNA coated gold particles integrates into plant chro.

Question 30

What is a knockout mouse

Answer 30

lab mouse where researchers have inactivated an existing gene by replacing it or disrupting it with an artificial piece of DNA

Question 31

why are knockout mice useful

Answer 31

provide valuable clues about what that gene normally does. have been used to study cancer, obesity, diabetes, arthritis etc

Question 32

Disadvantages of knockout mice

Answer 32

-15% of altered embryos cannot grow into adult mice -knocking out a gene may fail to produce an observable change

Question 33

RNA splicing

Answer 33

-pre-mrna formed at the end of transcription -contains both introns and exons -introns are removed -exons are spliced together -mature mRNA is then translated to produce ppc

Question 34

Alternative splicing

Answer 34

-some introns retained within mature mRNA and exons can be joined in different combinations -1 gene can code for many proteins

Question 35

advantages of alternative splicing

Answer 35

increases genetic diversity

Question 36

purpose of RNA interference (iRNA)

Answer 36

control gene expression ie. can silence genes

Question 37

Small interference RNA (siRNA) vs microinteference RNA (miRNA)

Answer 37

siRNA: must be fully complementary to mrna more precise double stranded miRNA: binds in many places in mRNA so only requires partial complimentary pairing less precise single stranded

Question 38

How iRNA works

Answer 38

-double-stranded iRNA binds to dicer(endonuclease) -dicer cuts iRNA into short sections -short iRNA binds to argonaute protein -1 strand of iRNA (guide strand) remains bound to argonaute protein to form RISC(RNA-induced-silencing-complex) -RISC complex binds* to mature mRNA -prevents translation of mRNA *cleavage catalysed by argonaute protein

Question 39

Gene therapy process

Answer 39

-adds functional therapeutic genes to cells with defective genes within them -allows genetic disorders to be treated

Question 40

2 main problems with gene therapy

Answer 40

-getting functional genes into cell -making functional genes work once they're in the cell

Question 41

Severe combined immunodeficiency disease (SCID) causes

Answer 41

Faulty ADA allele -ADA essential for proper functioning of immune system so no cell mediated response etc

Question 42

Delivery methods for functional therapeutic genes (FTG)

Answer 42

-Viruses eg. adenovirus -Liposomes i.e. FTG wrapped in lipid molecules. Then, sprayed into nostrils, cross csm and enter cells of the lung both ex vivo delivery methods

Question 43

Difficulties with viral vectors

Answer 43

-difficult to control where the virus inserts the gene -if gene inserted into gene that controls cell division, it can cause cancers

Question 44

Germline gene therapy

Answer 44

adds to FTG into fertilised ovum ensures all cells of organism will contain the FTG so FTG can be passed on to next generation

Question 45

Why are stem cells ideal for gene therapy

Answer 45

-unspecialised -divide by mitosis to form clones -pluripotent, so stem cells will develop into any tissue required

Question 46

Ethical complications of germline

Answer 46

-Playing as god -negative effects on future generations -ethical debate- changing genomes of unborn children -abuse of principle- designer babies?