21. recombinant DNA technology

Question 1

Five stages of DNA Technology

Answer 1

• isolation of DNA for a specific protein (creation of DNA fragment)
• insertion of DNA into a vector
• transformation of DNA into a host
• identification of successful hosts
• cloning/growth of host cells

Question 2

Describe how reverse transcriptase can be used to isolate a DNA fragment.

Answer 2

Enzyme that uses mRNA as template and joins DNA nucleotides, resulting in single-stranded cDNA,
+ DNA polymerase = DNA (H-bonds)

Question 3

What are three way that DNA fragments can be isolated?

Answer 3

Reverse transcription
Restriction endonuclease
Gene machine

Question 4

Describe how a gene machine can be used to create DNA fragments

Answer 4

Machine that works out mRNA and DNA sequences from amino acids.
Sequence is checked for biosafety and biosecurity, ensuring that gene is safe and ethical to produce
Results in oligonucleotides

Question 5

What are oligonucleotides and what makes them?

Answer 5

small sections of overlapping, single stranded nucleotides
produced by gene machine

Question 6

Describe how restriction endonuclease can be used to produce a DNA fragment.

Answer 6

• recognition sites bind the enzyme to specific areas on DNA base sequence
• results in staggered/sticky ends

Question 7

Give an advantage and disadvantage of using reverse transcriptase.

Answer 7

Advantage
- lots of mRNA of interest form selected cell

Disadvantage
- time consuming

Question 8

Give an advantage and disadvantage of restriction endonuclease.

Answer 8

Advantage
- sticky ends make insertion easier

Disadvantage
- still contains introns

Question 9

Give an advantage and disadvantage of gene machine.

Answer 9

Advantage
- quickly makes exact fragments of DNA with sticky ends

Disadvantage
- need to know sequence of amino acids beforehand

Question 10

Why might host cells not get transformed?

Answer 10

• Plasmid rejoins before DNA fragment could bind
• Plasmid does not enter host cell

Question 11

What is the difference between in vivo and vitro cloning?

Answer 11

In vivo involves use of a vector to clone recombinant DNA
in vitro (PCR) doesn’t.

Question 12

Describe how DNA is inserted into vector.

Answer 12

• plasmid inside vector is cut using same reverse transcriptase that cut DNA fragment.
• results in complementary sticky ends between the DNA fragments.
• the enzyme ligase aneals them together. (= phosphodiester bonds)

Question 13

What is a plasmid?

Answer 13

Circular loop of DNA, found in bacteria, separate from rest of genome

Question 14

How is the host cell ‘transformed’ with a vector? (in vivo cloning)

Answer 14

• membrane of host cell is made more permeable by mixing with Ca+ and heat shocked
• vector can now diffuse across membrane

Question 15

What are three types of marker genes that can be used to identify transformed host cells?

Answer 15

• antibiotic resistance genes
• fluorescent gene markers
• Enzyme markers.

Question 16

How are DNA fragments (made by restriction endonuclease) modified?

Answer 16

Promoter region added to the start of DNA. This is the binding site for RNA polymerase to enable transcription

Terminator region added to the end of gene. causes RNA polymerase to detach, ending transcription between genes

Question 17

Explain how antibiotic-resistance genes can be used to identify transformed cells.

Answer 17

Plasmid has genes for the resistance of tetracycline and ampicillin.

Insertion of DNA fragment disrupts resistance to tetracycline.

Use a sterile stamping block to transfer colonies of bacteria onto agar plate with ampicillin.

repeat onto tetracycline

cultures that grow on ampicillin have successfully taken in plasmid, cultures that don’t grow on tetracycline have taken up DNA fragment. (=clone from original plate)

Question 18

How can fluorescent markers be used to identify transformed cells?

Answer 18

green fluorescent protein (GFP) gene inserted to plasmid.
DNA fragment disrupts GFP gene and prevents production
non-glowing/ fluorescent colonies contain recombinant plasmids

Question 19

How can enzyme markers be used to identify transformed cell.

Answer 19

The gene for the enzyme lactase inserted into plasmid, disrupted by DNA fragment.
colonies grown on colourless substance (blue if lactase gene not disrupted)

Question 20

Explain the PCR method.

Answer 20

The temperature increased to 95C to break H bonds between DNA.

temperature is then decreased to 55C, so primers can attach (annealing)

taq DNA polymerase then attaches free nucleotides and makes new strand. (synthesis) the temperature is increased to 72 (optimal for taq DNA polymerase.

Question 21

three advantages of PCR

Answer 21

automated, efficient
Rapid,
no living cells, less complex + quicker

Question 22

difference betwen taq polymerase and normal DNA polymerase

Answer 22

bacterial protein that can withstand high temperatures (used in PCR)

Question 23

what are DNA probes?
what are they used for?

Answer 23

labeled single-stranded pieces of DNA (fluresc or radio)
locate specific gene location and screen for heritable conditions

Question 24

How are DNA probes used?

Answer 24

sample of patient DNA is heated to make it single-stranded
it is mixed with DNA proves that are complementary to a range of genes and alleles (eg for diseases)
if patient has that allele, DNA will bind with this probe, identified with x-ray or UV

…or a specific allele (for specific disease)

Question 25

what is DNA hybridisation?
How is this used?

Answer 25

DNA is heated to separate double helix into single strands. mixed with complementary single stranded DNA

once cooled, complementary strands anneal.

find particular allele (with DNA probes) used to select medicines based on genotypes. (some drugs more useful dependant on genotype)

Question 26

what is genetic counselling?

Answer 26

advice based on screening results of disease causing alleles.
eg lifestyle, monitoring.

Question 27

what are VNTRs
how can they be used?

Answer 27

variable tandem repeats, repeated sequences in introns. closely related = similar VNTRs.

genetic fingerprinting studies VNTRs, to determine genetic relationships and variability.

Question 28

Briefley explain 7 steps of genetic fingerprinting,

Collection/extraction, digestion, seperation, hybridisation, development, analysis

Answer 28

Collection
eg crime scene

Extraction
PCR (if sample size is small)

Digestion,
restriction endonuclease cuts out VNTRs

Separation, (gel electrophoresis)
placed on polar gel negative DNA moves to the positive end of gen. smaller pieces travel further due to resistance of gel.

Hybridisation,
DNA probes added, complementary to VNTr

Development
probes transferred to nylon sheet, were they can be exposed to UV or X-ray

Analysis

Question 29

How can DNA fingerprinting bands be interpreted?

when might you use it?

Answer 29

bands compared to different organisms, most matching/ similar bands are more likely to be unknown sample

paternity, crime scene, medical diagnosis, avoid inbreeding

Question 30

define genome and phenome

Answer 30

genome is complete set of genes in cell, phenome is all the proteins that a cell is able to produce