chapter 21

Question 1

recombinant DNA technology

Answer 1

combining different organisms DNA to enable scientists to modify and alter DNA to better industrial processes and medical treatments.

Question 2

creating DNA fragments using reverse transcription

Answer 2

uses reverse transcriptase enzmye
occurs naturally in viruses e.g. HIV

1) reverse transcriptase enzyme converts RNA into DNA
2) select a cell with the desired protein
3) this cell will contain lots of mRNA copies for the protein.
4) reverse transcriptase enzyme joins DNA nucleotides with complimentary bases to mRNA sequence
5) single stranded DNA fragment produced
6) DNA fragment made double stranded by adding DNA polymerase enzyme

a cell only has two copies of each gene so difficult to access.

Question 3

what is the advantage of using reverse transcription?

Answer 3

cDNA/single stranded DNA has no intorns as made from mRNA sequence

Question 4

restriction endonucleases

Answer 4

cut DNA at palindromic sites called restriction sites
enzymes which cut up the DNA
occur naturally in bacteria as defence mechanism

different restriction endonuclease enzymes which have active ssites complimentary to different DNA base sequences
this is scalled the recognition sequence
enzymes cut DNA at specific location

some enzzzymes cut double stranded DNA at the same point
this creates blunt ends

some enzymes create exposed DNA bases and staggered ends

these staggered ends are palindromic- read the same forwards and backwards
referred to as stick ends as they can join DNA with complimentary base pairs.

Question 5

gene machine

Answer 5

creating DNA fragments in the lba using a computerised machine.

examine protein of interest to identify amino acid sequence so the DNA and mRNA sequence can be identified

DNA is put into computer to check biosafety and biosecurity- to check that the DNA being produced is safe and ethical

computer can then make small sections of single stranded overlapping nucleotides that make up the gene- called oligonucleotides.

oligonucleotides are then joined together to create DNA for gene.

this method is
quick
accurate
produces intron free DNA
which can then be transcribed by prokaryotes.

Question 6

pros and cons of using reverse transcription

Answer 6

pros- mRNA in cell actively transcribed from genes so lots of mRNA to make single stranded DNA

cons- many steps so more time consuming
technically more difficult.

Question 7

pros and cons of using restriction endonucleases

Answer 7

pros- sticky ends of DNA fragments make it more easy to insert to create recombinant DNA

cons- still contains introns

Question 8

pros and cons of using gene machine

Answer 8

pros- can design exact DNA fragments with labels, sticky ends and preferntial codons

cons- need to know amino acid/base sequence.

Question 9

how are the DNA fragments modified and why?

Answer 9

DNA fragments need to be modified to ensure transcription of genes occurs

promoter region added to start of DNA fragment
promoter is a sequence of bases which RNA polymerase binds to, to initiate transcription.

Question 10

terminator

Answer 10

added at the end of the gene
causes RNA polymerase enzyme to dettach and stops transcription.
so only one gene is transcribed at a time.

Question 11

vectors

Answer 11

carry isolated DNA into host cell
most common is plasmids
-circular DNA
-separate from bacterial genome
-only a few genes.

Question 12

inserting the DNA into the vector

Answer 12

plasmid cut open using restriction endonucleases to create sticky ends

so sticky ends of DNA fragments are complimentary to sticky ends of plasmids.

Question 13

DNA ligase enzyme

Answer 13

cut plasmids and DNA fragments are joined together by DNA ligase enzymes
catalyses formation of phosphodiester bonds between nucleotides.

Question 14

transformation

Answer 14

for the vector/plasmid with recombinant DNA to be inserted into the host cell, the host cell membrane’s permeability needs to be increased

-this is done by mixing it with calcium ions and heat shocking it/suddenly increasing temperature.

so now the vector can enter the host cell’s cytoplasm.

Question 15

amplifying DNA fragments

Answer 15

once the DNA fragments have been isolated, they need to be cloned to create large quantiies
can be done in vivo or in vitro.

DNA can be amplified in vitro using PCR/polymerase chain reaction
this is done using an automated machine.

Question 16

equipment used in PCR

Answer 16

-DNA polymerase enzyme- taq polymerase- found in bacteria in hot springs- optimum temperature is much higher/72 degrees/does not denature at high tempoeratures
-thermocyle- machine
-DNA nucleotides
-primer- short sequence of DNA complimentary to start of DNA sample.

Question 17

3 stages of the PCR

Answer 17

denaturation
annealing
synthesis

Question 18

PCR

Answer 18

DNA is heated to 95 degrees to break hydrogen bonds between complimentary bases- separating two strands- denaturation
DNA is cooled to 55 degrees so the primer can attach- annealing

snyhesis- DNA polymerase attaches complimentary free nucleotides and makes a new strand to align next to each template

DNA is heated to 72 degrees as this is optimum temperature for taq dna polymerase.

Question 19

advantages of PCR

Answer 19

automated- so more efficient
rapid- 100B copies of DNA can be made within a few hours
doesn’t require living cells- less complex technology needed and rapid.

Question 20

what are DNA probes?
what are they used for?

Answer 20

DNA probes are short single stranded pieces of DNA with labelled radioactivity or flurorescene.
used to locate specific allele on a gene
screen patients for drug response, heritable conditions and health risks

Question 21

how is the DNA probe designed?
how is the patient’s DNA altered?

Answer 21

The DNA probe is designed with a complimentary base sequence to the allele being screened for.

The patient’s DNA is treated to make it single stranded and then it is mixed with the DNA probe.

Question 22

denaturation and annealing

Answer 22

the patient’s DNA sample is heated to break the hydrogen bonds between complimentary bases and make it single stranded- denaturation

single stranded patient DNA is added to DNA probe
some of the patient’s DNA will anneal to the DNA but some single stranded DNA will anneal to the DNA probe.

Question 23

how a specific allele is located

Answer 23

to locate a specific allele you need to know the DNA base sequence to create the DNA probe

you find the DNA sequence using DNA sequencing methods e.g. the sanger method

single stranded DNA fragment created using gene machine.

label is added e.g. radioactive nucleotide containing P32 isotope is added/ fluorescent label which emits light under UV light.

once hybridisation has occured, DNA is washed to remove any unbound DNA probe.

the presence of radioactive isotopes/fluroscence indicates that the allele you are testing for is present within the patient’s sample.

Question 24

genetic screening

Answer 24

DNA probes can be used to screen for genetic disorders or cancer causing oncogenes

you can screen for multiple genetic disorders at the same time by using an array which has multiple DNA probes attached.

Question 25

personalised medicine

Answer 25

one of the advantages of having your DNA screened

some medicines e.g. painkillers are more or less effective depeneding on your genotype.

-most effective dosages can be identified
-more effective, safe and reduced cost

e.g. vitamin E can be given to diabetic patients to reduce their risk of cardiovascular heart disease.
can increase the risk for other diabetic patients.

Question 26

genetic counselling

Answer 26

type of social work
people choose to have their family history reserached to see likelihood of them carrying alleles linked to diseases before startinga family or for their general health.

if they carried the allele, they would be informed of any future risks to themselves or their offspring.

Question 27

what diseases can be screened for?

Answer 27

cystic fibrosis
sickle cell anaemia
before starting a family

if there is a family history of breast cancer, you can be screened for alleles linked to this cancer.
if the allele is found, you can be screened more regularly for tumours, reduce environmental risk factors or have a masectomy.

Question 28

DNA probe

Answer 28

short sequence of DNA complimentary to specific allele/mutation/gene.

DNA probes are used to test for the presence of this allele/mutation/gene within the DNA sample

if the sequence is present, the DNA probe will hybridise/form hydrogen bonds and bind to the sequence.

wash to remove any unhybridised DNA probe
use labels e.g. UV light.
DNA must be single stranded for probe to hybridise.

Question 29

VNTR’s

Answer 29

95% of of human DNA is introns
consists of variable number tandem repeats

very unlikely that two individuals will have the same VNTR
the more similar their VNTR’s- the more closely related the two indiiduals are

gel electrophersis used to look at genetic variation within a population and genetic relationship between organisms.

Question 30

where are samples obtained from?

Answer 30

very small samples can be used from
body cells
hair folicless
blood

DNA fragments can be amplified using PCR to create large quantities.

use restriction endonuclease to cut DNA into smaller fragments
use enzyme which cuts very close to VNTR

add DNA fragments to small well containing agar gel
add liquid buffer and apply voltage.

DNA is negatively charged so moves towards positive end.
agar gel provides resistance
so smaller DNA pieces move further away and faster.
this is how different lengths of DNA are separated.

Question 31

how DNA probes are used in gel electrophersis

Answer 31

DNA probes- short sequence of DNA complimentary to specific VNTR
agar gel dries and cracks so DNA probe and VNTR moved to nylon cloth.

if VNTR present, DNA probe will hybridise
wash to remove any unbound DNA probe

view with labels e.g. UV light if fluorescent label was used or
use X ray to find position of VNTR

Question 32

uses of VNTR

Answer 32

paternity tests- VNTR of child will match up with VNTR of mother and father

compare DNA bands to
identify genetic relationships
compare sample with evidence from crime scene
identify presence gene linked to disease