25 Gene Technologies

Question 1

what is the function of PCR?

Answer 1

{polymerase chain reaction}

artificial DNA replication

Question 2

what are the four items required for PCR?

Answer 2

• the DNA fragment being copied
• free phosphorylated nucleotides - to synthesise new DNA strand
• Taq. DNA polymerase - to bind nucleotides together
• primers - provide starting sequence for Taq. DNA polymerase

Question 3

what is the name and function of the computer used in PCR?

Answer 3

a thermocycler

varies temperature at precise time intervals

Question 4

PCR: strand separation (stage 1)

Answer 4

target DNA heated to 95.C for 5 mins –> separated into 2 single strand lengths

Question 5

PCR: primer binding (stage 2)

Answer 5

solution cooled to 55.C

primers H bond to complementary base sequences on single stranded DNA

Question 6

PCR: strand synthesis (stage 3)

Answer 6

heated to 72.C (optimum for Taq. DNA polymerase)

free complementary nucleotides joined to DNA strands, beginning at the primers –> 2 identical strands produced from original

Question 7

PCR v. semi-conservative DNA replication

Answer 7

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

• PCR produces only short strands ; whole chromosomes copied in S-C R
• PCR requires primers
• PCR requires high temperatures to separate DNA ; S-C R uses DNA helicase

Question 8

what is the function of electrophoresis?

Answer 8

preparation and analysis of DNA for sequencing

Question 9

what are the three items required for electrophoresis?

Answer 9

• gel plate containing agarose, and covered by electrolyte buffer
• electrodes
• DNA strands

Question 10

how are samples of DNA obtained in electrophoresis?

Answer 10

restriction endonucleases cut DNA into fragments

Question 11

how do the DNA fragments separate during electrophoresis?

Answer 11

placed in wells at cathode end of tray

current passed through for ~2 hours

phosphate groups are attracted to the anode

shorter fragments of DNA move further and faster

Question 12

how are the DNA bands visualised following electrophoresis?

Answer 12

using fluorescent dye and UV light

Question 13

electrophoresis v. chromatography

Answer 13

+ involves the displacement of different molecules within a medium for identification
+ separates molecules according to size
+ some molecules slowed more by stationary phase than others

Question 14

what are haplotypes?

Answer 14

sets of genes inherited together from one parents

Question 15

what types of DNA are used when identifying haplotypes? why?

Answer 15

mitochondrial and Y-chromosomal DNA

neither undergo recomibination

Question 16

what are single nucleotide polymorphims (SNPs)?

Answer 16

sequences of DNA that vary between individuals by a single nucleotide

Question 17

what is a modern use of identifying SNPs?

Answer 17

give and indication of susceptibility to disease

and responses to drugs/chemicals/vaccinations

(can also track inheritance)

Question 18

what are variable number tandem repeats (VNTRs)?

Answer 18

patterns of repeated adjacent nucleotides

Question 19

what is a modern day use of VNTRs?

Answer 19

use as genetic fingerprint

can be used in electrophoresis –> forensic analysis

Question 20

what is the definition of genetic engineering?

Answer 20

the manipulation of an organism’s DNA

Question 21

what is the differenced between ‘transformed’ and ‘transgenic’ organisms?

Answer 21

transformed = organisms with recombinant DNA

transgenic = organisms which have been GE to include a gene from a different species

Question 22

how do restriction enzymes (endonucleases) isolate a DNA fragment?

Answer 22

enzyme has specific recognition sites where it digests DNA

some produce a staggered cut (‘sticky ends’), which can anneal to other complementary sequences

Question 23

how is a DNA fragment inserted into a vector? (4 stages)

Answer 23

DNA ligase cuts vector DNA, then anneals plasmid and recombinant gene

bacteriophages are used to inject DNA into host cell

ice cold CaCl(2) encourages bacteria to take DNA up by increasing permeability of bacterial cell wall

plasmids added + mixture heat shocked to 45.C for 1-2 mins

Question 24

how is a gene produced from an mRNA template? (4 stages)

Answer 24

reverse transcriptase synthesises a single strand of cDNA complementary to mature mRNA

DNA polymerase used to join free nucleotides together to form second DNA strand

plasmid vector opened with restriction enzyme

gene placed in plasmid with DNA ligase

Question 25

why are reporter genes required in genetic engineering?

Answer 25

to identify which bacterial cells have taken up the recombinant DNA

Question 26

how do reporter genes work?

Answer 26

desired gene inserted into reporter gene sequence so that it does not work

cells not displaying reporter property (e.g. fluorescence) have taken up gene

Question 27

how are bacteria encouraged to take up recombinant DNA?

Answer 27

ice cold CaCl(2) - increases permeability of cell wall

growth on agar plates + replica plating

Question 28

describe the benefits of genetically engineering ‘Enviropig’

Answer 28

mouse + E. coli DNA prompts phytase production in pig’s salivary gland

phytase breaks down phosphorous into phosphates –> can be absorbed

∴ less phosphorous contaminates human water supply

Question 29

give an example of a genetically engineered crop plant

Answer 29

Agrobacterium tumefaciens

a naturally occurring soil bacterium which can introduce new genetic material into plant cells

infects wound sites of dicotyledonous plants, causing formation of crown gall tumours

Question 30

discuss ‘knockout mice’

Answer 30

mice that are GE to have an inactivated gene

target gene knocked out by incorporating a nucleotide sequence resembling it into an embryonic stem cell, which is then fused with a de-nucleated embryo

used to study effect of certain genes on diseases

Question 31

what is the difference between an intron and an exon?

Answer 31

exons code for sequences of amino acids

introns do not, and are removed from mRNA after transcription (–> mature mRNA)

Question 32

what is alternative splicing?

Answer 32

when mRNA molecules from the same gene have different combinations of introns retained and rejoined

Question 33

what is the function of alternative splicing?

Answer 33

allows genetic diversity

Question 34

give a use of RNA interference

Answer 34

gene control - post-transcriptional gene splicing

laboratory tool

treatment of disease

silencing of genes in tumour cells

reducing gene activity in cell culture

Question 35

what is the mechanism of RNA interference?

Answer 35

micro RNA (miRNA) and small interfering RNA (siRNA)

delivered from double-stranded RNA

dicer enzyme cuts double-stranded RNA into 20-25 nucleotide lengths

Argonaute protein selects and binds to one strand of RNA

siRNA binds to specific sequences of complementary nucleotides on mRNA

Argonaute protein breaks mRNA (making it non-functional)