Making DNA fragments Flashcards
What does recombinant DNA technology involve?
Transferring a fragment of DNA from one organism to another
How is recombinant DNA technology possible?
The genetic code is universal
Transcription and translation mechanisms are similar
What are the three ways you can produce DNA fragments?
- Reverse transcriptase
- Restriction endonuclease enzymes
- Gene machine
Why is it difficult to obtain a DNA fragment containing the target gene?
There are only a couple copies of each gene in each cell
Why does reverse transcriptase use mRNA?
Easier to obtain
Multiple copies of mRNA whereas only a couple copies of DNA
Why is mature mRNA used during the creation of DNA fragments using reverse transcriptase
There are no introns
They are non-coding
What does the mRNA act as during the creation of DNA fragments using reverse transcriptase?
a template strand for making DNA from
What does reverse transcriptase create from mRNA? How?
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
What do restriction endonuclease enzymes recognise?
Specific palindromic sequences of nucleotides
What are specific palindromic sequences also known as?
Recognition sequences
What is a palindromic sequence of nucleotides?
Base sequences which have antiparallel base pairs
Different restriction endonuclease enzymes…
Cut at different specific recognition sequences
What is possible if there are palindromic sequences at either side of the DNA fragment?
the restriction endonuclease is able to cut the fragment containing the target allele and separate it from the rest of the sequence
What does the restriction endonuclease enzymes sometimes leave once they cut the DNA fragment out?
Sticky ends which are small tails of unpaired bases
What is the purpose of sticky ends?
To anneal to other sticky ends in another piece of DNA if the two sticky ends are complimentary to each other
By what method does the restriction endonuclease enzyme remove the DNA fragment?
Hydrolysis
What is the method involving the use of a gene machine?
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
Explain the processes which take place to insert insulin from B-Cells in the pancreas into a vector. (6 marks)
- 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
