Recombinant DNA technologies

Question 1

3 ways of isolation

Answer 1

Reverse transcription
Restriction endonucleases
Gene machine

Question 1

3 ways of isolation

Answer 1

Reverse transcription
Restriction endonucleases
Gene machine

Question 2

Reverse transcription

Answer 2

Cell producing certain protein is selected
Cells have lots of mRNA for the protein
Reverse transcriptase binds complementary nucleotides to mRNA
Forms cDNA
DNA polymerase to make 2x strands
cDNA is intron free

Question 3

Restriction endonucleases

Answer 3

Enzymes that cut DNA
Enzyme has AS complementary to base sequences
Blunt + staggered cuts
Staggered cuts have sticky ends
Sticky ends are palindromic - same forwards as they are backwards

Question 4

Gene machine

Answer 4

Identify amino acid sequence and DNA sequence from proteins
Sequence entered into computer - checks safety and ethics
Computer creates small sections of overlapping single stranded nucleotides
-oligonucleotides
Can be joined to create DNA for entire gene
PCR used to amplify quantity
Quick, accurate and intron free

Question 5

5 steps of in-vivo cloning

Answer 5

Create fragments
Insertion
Transformation
Identify transformed cells
Grow host cell

Question 6

Insertion into vector

Answer 6

Cut plasmid with restriction endonuclease
Creates complementary sticky ends
Ligase binds DNA to plasmids

Question 7

Transformation of host cell

Answer 7

Vector inserted into host cell
CSM made more permeable to plasmids:
-mixed with Ca2+
-heat shocked
Allows vector to enter host cytoplasm

Question 8

Why do we need identification

Answer 8

Plasmid doesn’t enter the cell
Plasmid re joins before DNA enters
DNA fragment sticks to itself and not plasmid

Question 9

Antibiotic resistant markers

Answer 9

DNA inserted into vector into antibiotic resistant gene tetracycline, plasmid also contains ampicillin
Grow on agar
Stamp velvet on agar
Stamp onto new agar plate, genes not resistant to ampicillin die
Repeat on new agar
Genes not resistant to tetracycline die
Plasmids that survived ampicillin but died to tertracycline contain the DNA fragment

Question 10

Fluorescent markers

Answer 10

GFP in jelly fish
Fragment inserted
Non glowing cells contain fragment

Question 11

Enzyme markers

Answer 11

DNA inserted into lactase gene
Bacteria grown on agar plate with colourless substrate
Lactase turns colourless substrate blue

Question 12

In vitro

Answer 12

In non living thing

Question 13

What is PCR

Answer 13

Polymerase chain reaction

Used to amplify fragments from invitro

Question 14

PCR equipment list

Answer 14

Thermocycler
DNA fragments
Taq polymerase
Primers
DNA nucleotides

Question 15

Thermocycler process

Answer 15

Temp increase to 95 - breaks H bonds, split DNA into single strands
Temp decrease to 55 - primers can anneal to single strands
Temp increase to 72 - DNA polymerase attaches free nucleotides to single strands, optimum temperature for taq polymerase

Question 16

Advantages of PCR

Answer 16

Automated - more efficient
Rapid - 100 billion copies of DNA within hours
Doesn’t require living cells - quicker and less complex techniques needed

Question 17

DNA probes

Answer 17

Sample of patient DNA mixed with marked probes
If patient has allele, probe binds
Can be viewed by x-ray or UV light
Fluorescently or radioactively marked

Question 18

DNA hybridisation

Answer 18

DNA heated to separate strands
Mixed with complementary sequences
Used to identify certain alleles

Question 19

Personalised medicine and genetic counselling

Answer 19

Screening allows for medicine based on genotypes

Question 20

VNTRs

Answer 20

95% of DNA is introns
Low chance of people sharing same VNTRs
Genetic fingerprinting checks genetic relationships and variability in a population

Question 21

GFP - collection and extraction

Answer 21

Sample of DNA collected, amplified by PCR

Question 22

GFP - digestion

Answer 22

Restriction endonucleases added to cut close to VNTRs

Enzymes that cut close to target VNTRs are added

Question 23

GFP - separation

Answer 23

DNA samples loaded in small wells in agar gel
Gel placed in buffered liquid with voltage
DNA is negatively charged so moves toward positive end of gel
-electrogelphoresis, smaller pieces move further
-VNTRs separated
-alkaline used to separate double strands

Question 24

GFP - hybridisation

Answer 24

DNA probe process

Question 25

GFP - development

Answer 25

Agar shrinks and cracks, so moved to nylon sheet

Exposed to X-rays for radioactive probes, UV light for fluorescent probes

Question 26

GFP - analysis

Answer 26

Position of DNA bands are compared to identify genetic relations