Gene Technology

Question 1

What does recombinant DNA technology entail?

Using bacteria

Answer 1

Isolation of desired gene
Insertion into vector
Transformation into host
Identification with gene markers
Growth + cloning

Question 2

Why can DNA fragments be inserted into genome of other organisms?

Answer 2

Genetic code universal

Mechanisms involved in translation + transcription = universal

Question 3

What are the methods for gene identification?

Answer 3

mRNA to cDNA using reverse transcriptase

Question 4

Describe viral replication

Answer 4

HIV binds to T cells
Capsid fuses with membrane Reverse transcriptase converts RNA to DNA
DNA moves into nucleus to create mRNA
New viral proteins made
HIV breaks away from T cell

Question 5

Describe what happens in producing DNA fragments

Answer 5

Protein producing cell with large amounts of MRNA selected
Desired gene’s mRNA extracted
Reverse transcriptase forms single stranded complementary copy of DNA (cDNA) from mRNA
Hydrolysis of mRNA isolates cDNA
DNA polymerase forms DNA on template cDNA
Desired gene formed
Could be inserted into plasmids

Question 6

What is restriction endonuclease?

Answer 6

Restriction enzyme

Question 7

What does restriction endonuclease do?

Answer 7

Cuts DNA at specific sequence of bases

Producing either “blunt ends” or “sticky ends”

Question 8

Why are sticky ends useful?

Answer 8

Are a palindrome
Same forward + backwards
4 unpaired bases opposite
Complementary plasmid
Recognition sequence is 6bp palindromic sequence

Question 9

Explain why pieces of human DNA would be able to join to the cut DNA of plasmids

Answer 9

Sticky ends

Complementary base pairing

Question 10

Cut plasmids + lengths of foreign DNA can join. What features of their ends allow them to join?

Answer 10

Sticky ends

Complementary base pairing

Question 11

What is the gene machine?

Answer 11

Used to create gene required

Synthesis of DNA from scratch

Question 12

Why is the gene machine useful?

Answer 12

No need for pre-existing DNA template

Question 13

How is the gene machine used to produce DNA fragments?

Answer 13

DNA sequence designed + fed into computer
1st nucleotide fixed to support
Nucleotide added in correct order
Protecting groups added to ensure bases added at right place
Nucleotides added Computer designs oligonucleotides
Protecting groups removed + oligonucleotides joined
Double strands formed to replicate gene
Gene inserted into plasmid
Vector stores, replicates + transfers gene
New gene checked for mistakes
Bacteria carrying gene cloned

Question 14

In vivo amplification once DNA fragment containing desired gene you need is isolated you need to…

Answer 14

Clone it

Produce enough for medical or commercial use

Question 15

What are the two main methods?

Vivo amplification

Answer 15

In vivo = transferring into host cell using vector

In vitro = polymerase chain reaction

Question 16

Describe outline of vivo amplification?

Answer 16

Isolation
Insertion
Transformation
Identification
Growth

Question 17

What does isolation + insertion require in vivo amplification?

Answer 17

Restriction endonuclease
Complementary sticky ends
Vector
Plasmid

Question 18

Describe isolation + insertion

Answer 18

Plasmid cut open at desired gene recognition site using same restriction endonuclease
DNA fragment + plasmid sticky ends are complementary
Desired gene cut out using restriction endonuclease
DNA fragments + open plasmid mixed with DNA ligase
Plasmids contain new gene

Question 19

Explain why the same restriction endonuclease enzyme must be used (4 marks)

Answer 19

Cuts same base sequence
Ensures sticky ends are complementary
Only cuts required gene
Ensures DNA fragments can be inserted into plasmid

Question 20

For transcription to take place when preparing fragments what do you require?

Answer 20

RNA polymerase
Transcription factors to bind near gene
Promoter region = tell RNA polymerase where to bind
Terminator = releases RNA polymerase

Question 21

What happens in transformation?

Vivo amplification

Answer 21

Introduce recombinant DNA into host
Mixing bacterial cell + plasmid in medium
Medium ice cold + contains Ca2+

Question 22

Why is transformation in vivo amplification done in these conditions?
Ice cold + Ca2+

Answer 22

Ice cold chloride solution = increases permeability

Heat shock = encourages uptake

Question 23

What are the three methods for identification in vivo amplification?

Answer 23

All use 2nd separate gene on plasmid that is
Resistant to antibiotic
OR
Make fluorescent protein
OR
Produce enzyme whose actions can be identified