(21) recombinant DNA technology

Question 1

define genome

Answer 1

entire set of DNA including all the genes in an organism

Question 2

how is gene sequencing done

Answer 2

smaller fragments are sequenced first and then put back into order to give the whole genome

Question 3

define proteome

Answer 3

all the proteins made by an organism

Question 4

why is determining the proteome useful in medical research

Answer 4

identifying protein antigens on the surface of disease causing bacteria and viruses can help in the development of vaccines to prevent the disease

Question 5

what are regulatory genes

Answer 5

they determine when the genes that code for particular proteins should be switched on and off

Question 6

what is pyrosequencing

Answer 6

recently developed technique that can sequence around 400 million bases in a 10 hour period

Question 7

what is recombinant DNA technology

Answer 7

transferring a fragment of DNA from one organism to another because genetic code is universal and transcription and translation mechanisms are pretty similar, the trasnferred dna can be used to produce a protein in the recipient cells

Question 8

what are the 3 ways dna fragments can be produced

Answer 8

1) using reverse transcriptase
2) using restriction endonuclease enzyme
3) using a ‘gene machine’

Question 9

how can dna fragments be made using reverse transcriptase

Answer 9

mRNA can be used as templates to make lots of dna. reverse transcriptase then makes dna from an rna template. the dna made is called complementary dna (cDNA)

Question 10

how can dna fragments be made using restriction endonuclease enzyme

Answer 10

restriction endonuclease recognises palindromic sequences (recognitiion sites) and cuts the dna at these places. different enzyme used for different recognition sites as has to be complementary. dna sample is then incubated with specific restriction endonuclease which cuts dna fragment out by a hydrolysis reaction. this sometimes leaves sticky ends

Question 11

what can sticky ends be used for

Answer 11

to bind the dna fragment to another piece of dna that has sticky ends with complementary sequences.

Question 12

how can dna fragments be made using a ‘gene machine’

Answer 12

technology that synthesises dna fragments from scratch with no need for a pre existing dna template. database contains necessary info to produce the dna fragment. sequence is designed, first nucleotide is fixed to a bead and more nucleotides are added and protecting groups prevent unwanted branching. then broken off from bead and protecting groups are removed

Question 13

why do dna fragments need to be amplified

Answer 13

so we have sufficient quantity to work with

Question 14

outline the process of in vivo amplification

Answer 14

1) dna fragment inserted into vector (plasmid or bacteriophages). vector is cut open using same restriction endonuclease that was used to isolate fragment sticky ends of vector and dna are then complementary. vector dna and fragment dna are mixed together using dna ligase (new combination of DNA + DNA fragment is recombinant dna.)
2) vector transfers gene into cells (only about 5% of host cells will take up the vector)
3) marker genes can be used to identify the transformed genes

Question 15

how can you produce proteins coded for by the dna fragment using transformed host cells

Answer 15

vector must contain specific promoter and terminator regions.
promoter regions- dna sequences that tell the enzyme rna polymerase when to start producing mRNA and terminator regions tell it when to stop. without the right promoter region the DNA fragment wont be transcribed and protein wont be made

Question 16

what is in vitro cloning

Answer 16

when copies of a dna fragment are made outside of a living organism using the polymerase chain reaction.

Question 17

what are the stages of the polymerase chain reaction (in vitro cloning to make dna fragments)

Answer 17

1) reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase is heated to 95 to break H bonds between 2 dna strands. mixture cooled to 50-65 so primers can bind to strands
2) heated to 72 so dna polymerase can work- lines up free dna nucleotides alongside each template strand and complementary strands are formed.
3) 2 copies of dna fragment are formed in one PCR cycle

Question 18

what are transformed organisms and how can they be made

Answer 18

microorganisms, plants and animals can be transformed (genetic engineering). foreign dna can be inserted into:

1) microorganisms- to produce useful protein
2) plants
3) animals- desirable gene inserted into embryo or egg cell

Question 19

how can recombinant dna benefit the agriculture industry

Answer 19

tranformed to give higher yields or more nutritious - reduces risk of famine and malnutrition

Question 20

how can recombinant dna benefit the medicinal industry

Answer 20

many drugs and vaccines are produced from transformed organisms eg insulin for T1 diabetes used to come from animals but didn’t work that well for humans but transformed microorganisms can now use a cloned insulin gene to make insulin

Question 21

what are the concerns associated with using recombinant dna in agriculture

Answer 21

1) using only one type of transformed crop (monoculture) could make the whole crop vulnerable to disease also reduced biodiversity
2) superweeds could occur if transformed crops interbreed with wild plants- uncontrolled spread of recombinant dna with unknown consequences
3) organic farmers could have their crops contaminated with wind blown seeds from nearby GM crops so would no longer be organic- less income

Question 22

concerns of recombinant dna in medicine

Answer 22

1) companies who own genetic engineering technologies may limit the use of technologies that could be saving lives
2) could be used unethically e.g. designer babies (currently illegal)

Question 23

what are the ownership issues arisen from recombinant dna

Answer 23

1) who owns human genetic material once it has been removed from the body- donor or researcher?
2) large corporations own patents to particular seeds and charge high prices and can require farmers to repurchase every year. if non GM is contaminated by GM farmers can be sued

Question 24

what are the humanitarian benefits of recombinant dna technology

Answer 24

1) agricultural crops can be produced that reduce famine and malnutrition
2) transformed crops can be used to make useful pharmaceutical products so can be available for more people
3) medicine can be made more cheaply so more can afford it
4) potential to be used in gene therapy to treat human disease

Question 25

how does gene therapy work

Answer 25

1) altering defective genes to treat people with cancer and genetic disorders. if disorder is caused by recessive alleles you can add a working dominant one to make up for them. if its caused by a mutated dominant allele you can silence it by sticking a bit of dna in the middle.
the allele / dna is inserted using vectors

Question 26

what are the 2 types of gene therapy

Answer 26

somatic therapy- altering alleles in human body but doesn’t affect sex cells so offspring could still inherit disorder
germ line therapy- altering alleles in sex cells- any offspring wont inherit disorder (illegal in humans)

Question 27

what are dna probes

Answer 27

short strands of dna with a specific base seq complementary to base seq of target allele so will bind to target allele. used for locating certain alleles
probe has a label attached so it can be detected (radioactive or fluorescent)

Question 28

how do dna probes identify alleles

Answer 28

sample of dna is digested into fragments using restriction enzymes and separated using electrophoresis. fragments transferred to nylon membrane and incubated with dna probe which will bind to target allele. then exposed to uv light and if gene is present there will be a fluorescent band

Question 29

how can a probe be used as part of a dna microarray

Answer 29

dna microarray is glass slide with spots of different dna probes attached in rows. fluorescently labelled human dna is washed over array any complementary probes will then stick. array is washed to remove any unstuck dna. array is then shown under UV to show up any attached labelled dna

Question 30

how do you produce a dna probe

Answer 30

seq allele that you want to screen for and use PCR to produce multiple complemetary copies of the part of the allele- these copies are the probes

Question 31

what is genetic counselling

Answer 31

advising patients and relatives about the risks of genetic disorders eg explaining results of screenings , advising on prevention or treatment available

Question 32

what is personalised medicine

Answer 32

medication that are tailored to an individuals dna so you are prescribed the ones that are most effective for you

Question 33

what are variable number tandem repeats

Answer 33

base seq that don’t code for proteins and repeat next to each other (sometimes thousands of times)

Question 34

why do no 2 individuals have the same genetic fingerprint

Answer 34

because the probability of 2 individuals having the same number of VNTRs at each place they’re found in dna is very low

Question 35

how is a genetic fingerprint made

Answer 35

1) sample of dna obtained eg blood, saliva
2) PCR makes many copies of the areas of DNA that contain the VNTRs
3) fluorescent tag added to all DNA fragments so they can be viewed under UV light
4) dna fragments undergo electrophoresis: dna mixture covered in buffer sol that conducts elec, current passes through and dna fragments move towards pos electrode. dna fragments separate according to size as smaller move faster
5) dna fragments viewed as bands under UV light (genetic fingerprint)

Question 36

what are the uses of genetic fingerprinting

Answer 36

1) determining genetic relationships

2) determining genetic variability within population