Manipultion of DNA Flashcards
How does recombinant DNA technology work?
It is gene cloning that requires isolating sections of DNA and cloning them to make multiple copies
In recombinant DNA technology small pieces of DNA is required.
What enzyme is used to achieve this?
How does they work?
Restriction enzymes
They recognise specific nucleotide sequences and act as molecular scissors to cut the DNA into smaller sections
What is the difference between blunt or sticky ends?
When restrictive enzymes cut DNA they can either leave both strands flush (blunt) or angled (sticky)
Are blunt or sticky ends best for cloning?
Sticky
How can the newly cut DNA fragments be cloned?
What molecule helps with this process?
They are inserted into plasmids
What is a plasmid?
A small circular strand of DNA found in bacteria which can replicate independently of chromosomes
How does DNA fit into a plasmid?
The same restriction enzymes which cut the DNA also cut the plasmid, allowing a complementary fit to form
What is the complex called that consists of a DNA fragment and a cut plasmid?
A recombinant DNA molecule
Which enzyme joins the DNA to the cut plasmid?
DNA ligase
How is a recombinant DNA molecule cloned?
It is taken up by bacteria which use the origin of replication to produce 100-200 copies
What type of DNA is used for the recombinant DNA process for eukaryotic genes?
Mature mRNA as there is not process in bacteria to splice exons and remove introns
The mature mRNA is then converted back to DNA
What is the name for DNA which has been produced from mature mRNA?
Complimentary DNA
cDNA
Which enzyme is responsible for producing cDNA from mature mRNA?
Reverse transcriptase
What is different about cDNA for regular DNA?
It is exons only, as the introns have been removed during the mature mRNA formation
What is the difference between deoxynucleotides and dideoxynucleotides?
Deoxynucleotides have an OH at carbon 3 but dideoxynucleotides jut have a H
What are ddNTP used for?
How does this process work?
For gene sequencing to identify the sequence of base pairs, as the ddNTP interrupt the DNA polymerase enzyme from extending the sequence, therefore where the sequence ends must be the base of the ddNTP in the mix
E.g. ddATP (adenosine)
How is automated DNA sequencing one step above dideoxynucleotide DNA sequencing?
It uses all x4 ddNTP’s at once and they are each marked with a fluorescent marker
What is PCR?
What does it do?
Polymerase chain reaction
It is another way of making clones of DNA fragments
How does PCR work?
What are it’s X3 steps?
Can you name some primers?
How long does each step take?
What does the temperature need to be for each step?
It uses primers to recognise the sequences of DNA at the ends of small DNA fragments
The steps are:
1) denaturation
= slitting the DNA into single strands at a heat of 95 degrees
= 30 seconds
2) primer annealing
= reducing the temperature to around 45-68 degrees to allow primers (TAQ or PFU) to hybridise
= 30 seconds
3) primer extension
= temperature increased to 72 degrees
= TAQ polymerase extends the primers to synthesise DNA
= 60 seconds
