Making DNA fragments

Question 1

What does recombinant DNA technology involve?

Answer 1

Transferring a fragment of DNA from one organism to another

Question 2

How is recombinant DNA technology possible?

Answer 2

The genetic code is universal

Transcription and translation mechanisms are similar

Question 3

What are the three ways you can produce DNA fragments?

Answer 3

• Reverse transcriptase
• Restriction endonuclease enzymes
• Gene machine

Question 4

Why is it difficult to obtain a DNA fragment containing the target gene?

Answer 4

There are only a couple copies of each gene in each cell

Question 5

Why does reverse transcriptase use mRNA?

Answer 5

Easier to obtain

Multiple copies of mRNA whereas only a couple copies of DNA

Question 6

Why is mature mRNA used during the creation of DNA fragments using reverse transcriptase

Answer 6

There are no introns

They are non-coding

Question 7

What does the mRNA act as during the creation of DNA fragments using reverse transcriptase?

Answer 7

a template strand for making DNA from

Question 8

What does reverse transcriptase create from mRNA? How?

Answer 8

cDNA (complimentary DNA)
The mRNA is mixed with reverse transcriptase and DNA nucleotides
Reverse transcriptase adds complimentary DNA nucleotides to the mRNA template strand to create a DNA fragment

Question 9

What do restriction endonuclease enzymes recognise?

Answer 9

Specific palindromic sequences of nucleotides

Question 10

What are specific palindromic sequences also known as?

Answer 10

Recognition sequences

Question 11

What is a palindromic sequence of nucleotides?

Answer 11

Base sequences which have antiparallel base pairs

Question 12

Different restriction endonuclease enzymes…

Answer 12

Cut at different specific recognition sequences

Question 13

What is possible if there are palindromic sequences at either side of the DNA fragment?

Answer 13

the restriction endonuclease is able to cut the fragment containing the target allele and separate it from the rest of the sequence

Question 14

What does the restriction endonuclease enzymes sometimes leave once they cut the DNA fragment out?

Answer 14

Sticky ends which are small tails of unpaired bases

Question 15

What is the purpose of sticky ends?

Answer 15

To anneal to other sticky ends in another piece of DNA if the two sticky ends are complimentary to each other

Question 16

By what method does the restriction endonuclease enzyme remove the DNA fragment?

Answer 16

Hydrolysis

Question 17

What is the method involving the use of a gene machine?

Answer 17

DNA is synthesised from scratch
1 ) Sequence is designed
2) The first nucleotide is added to some sort of support eg. a bead
3) Nucleotides are added one by one in the right order in a series of processes, including adding protecting groups which make sure that the nucleotides are added in the right order
4) Short sections of DNA called oglionucleotides are produced. Once complete, they are broken off from the support and protecting groups are removed.
5) Oglionucleotides are joined together to make longer DNA fragments

Question 18

Explain the processes which take place to insert insulin from B-Cells in the pancreas into a vector. (6 marks)

Answer 18

• Reverse transcriptase uses mRNA from insulin as a template to produce cDNA (complimentary DNA)
• The single strand of cDNA is converted to a double strand of DNA containing the insulin gene
• Using DNA polymerase
• Sticky ends added to either side of the gene
• Copies made using PCR
• Plasmids are extracted from bacteria
• Plasmid is cut using restriction endonuclease which leaves a sticky end that is complimentary to those on the insulin gene
• DNA ligase joins the insulin gene into the plasmid which acts as a vector