6.3 Manipulating Genomes

Question 1

Steps of PCR

Answer 1

solution with DNA sample, DNA polymerase, primers and free nucleotides
95 degrees
cooled to 50-65 to anneal primers
72 degrees DNA polymerase

Question 2

describe gel electrophoresis

Answer 2

wells at negative end
agarose gel
buffer solution
loading dye
micropipette

Question 3

what must be done to proteins before gel electrophoresis?

Answer 3

mixed with a chemical that denatures the proteins to the same charge (as proteins are positively and negatively charged)

Question 4

what are protein electrophoresis used for?

Answer 4

present in urine and blood, used to diagnose disease

Question 5

what is a palindrome sequence?

Answer 5

a section of DNA that the bases are in the same order read backwards on the opposite strand e.g
GAATTC
CTTAAG

Question 6

what enzymes recognize palindromic sequences?

Answer 6

restriction enzymes

Question 7

what is used in DNA profiling?

Answer 7

gel electrophoresis using repeating non-coding units of DNA (very specific to each person) - tandem repeats

Question 8

what can DNA profiling also be used for?

Answer 8

risk for genetic disorders (e. embryo screening for cystic fibrosis)

Question 9

what are the 3 steps of genetic engineering?

Answer 9

1. desired gene obtained
2. inserted into vector
3. vector transfers gene into bacteria

Question 10

what is used in step 1 of genetic engineering?

Answer 10

using restriction enzymes

Question 11

what are two types of vectors?

Answer 11

plasmid (circular molecules of bacterial DNA)
bacteriophage (virus)

Question 12

what enzyme is used during mixing of DNA fragments and vector?

Answer 12

DNA ligase

Question 13

what happens in the process of ligation?

Answer 13

sugar-phosphate backbones

Question 14

what is the new DNA called in step 2 of genetic engineering?

Answer 14

recombinant DNA

Question 15

what process has to be done for plasmid vector to be taken in?

Answer 15

processes such as electroporation, uses an electroporator and electric field to increase membrane permeability

Question 16

how does a bacteriophage transfer DNA?

Answer 16

DNA is injected into the bacteria and the phage DNA integrates with bacterial DNA

Question 17

what is a genetically modified organism called?

Answer 17

transgenic organism

Question 18

where does the gene come from that is inserted into soy beans?

Answer 18

bacillus thuringiensis (Bt)

Question 19

what bacteria is the desired gene for soy beans injected into?

Answer 19

Agrobacterium tumefaciens

Question 20

what is monoculture? why is it a problem?

Answer 20

1. growing only one type of crop
2. reduces genetic diversity making population more vulnerable to disease

Question 21

how is genetic engineering used for medicinal purposes?

Answer 21

GM to produce wanted products such as antithrombin in goats milk

Question 22

how can pathogens be used in research?

Answer 22

GM - e.g poliovirus can attack cancer cells, GM to not cause disease but only attack tumour cells

Question 23

what problems are associated with pathogens in research?

Answer 23

• fear of mass infection/outbreak
• GM pathogen may revert to its initial state and cause outbreak
• malicious use for biowarfare

Question 24

define gene therapy

Answer 24

the use of altering alleles inside cells to cure genetic disorders

Question 25

If a disorder is caused by two recessive alleles you can...?

Answer 25

add a dominant allele

Question 26

If a disorder is caused by a dominant allele you can...?

Answer 26

'silence' the gene by adding DNA in the gene that downregulates the gene

Question 27

what vectors are used in gene therapy?

Answer 27

viruses, plasmids or liposomes

Question 28

what are the two types of gene therapy?

Answer 28

somatic and germ line therapy

Question 29

what is somatic therapy?

Answer 29

altering alleles most affected by the disorder (e.g cystic fibrosis targets epithelial cells in the lungs) note: does not affect sex cells so any offspring could inherit

Question 30

what is germ line therapy?

Answer 30

alters alleles in sex cells. This means all cells are affected by gene therapy in offspring and won't inherit the disease but note: this is currently illegal

Question 31

What are the positive ethical implications of gene therapy?

Answer 31

- prolongs lives - improved QOL - decrease genetic disorders

Question 32

what are the negative ethical implications of gene therapy?

Answer 32

- risk of more harm (e.g overexpression of genes) - expensive (resources better spent elsewhere) - effects short lived (somatic) - immune response to vectors - allele inserted in the wrong places

Question 33

what is the chain termination method?

Answer 33

determines order of bases using modified stopping nucleotides

Question 34

what is in the mixture that starts the CTM?

Answer 34

single strand DNA template DNA primer DNA polymerase free nucleotides

Question 35

process of chain termination method?

Answer 35

PCR makes differing lengths of DNA which undergo gel electrophoresis which produces a complementary sequence which can be translated to the original sequence

Question 36

how long can chain termination reaction sequence?

Answer 36

up to 750bp

Question 37

what is done in pyrosequencing?

Answer 37

- DNA cut into fragments and attach to beads - amplified with PCR - each bead into a well - free nucleotides in wells - wells have enzymes that produce light when added to strands -computers analyses the light occurrence and intensities and interprets DNA sequence