Manipulating genomes Flashcards
What is DNA sequencing?
The process of working out the sequence of nucleotides within DNA.
What are the ingredients for DNA sequencing?
DNA for sequencing.
Primer
DNA polymerase
Excess of normal nucleotides
Terminator bases labelled with radioactive/fluorescent tags.
What do the terminator bases do?
They attach where a normal nucleotide would and stop DNA synthesis when they are included.
What do you need in each test tube for Sanger sequencing?
4 test tubes.
In each you need ;
The DNA being sequenced
A mixture of normal nucleotides in excess
One type of terminator nucleotide (ATC or G)
A primer
DNA polymerase
How can you then sequence the DNA in Sanger sequencing after the test tubes have produced lots of strands of DNA?
Eventually you will have built up all different lengths of DNA across the 4 test tubes.
If we visualise (gel electrophoresis) the fragments and line them up in order of length you can read the sequence from the terminator bases.
What is high-throughput sequencing?
A variety of approaches used to develop fast, cheap methods to sequence genomes.
Example is pyrosequencing.
What is PCR?
Polymerase Chain Reaction.
It can be described as the in vitro method of DNA sequencing.
What is PCR used for?
It is used to produce large quantities of specific fragments of DNA and RNA from very small quantities (even just one molecule of DNA or RNA).
Using PCR scientists can produce billions of identical copies of the DNA or RNA samples within a few hours, these can then be used for analysis.
How much DNA does PCR produce each cycle?
It doubles the DNA.
What is needed for PCR?
The target DNA being amplified.
Primers in excess.
DNA polymerase (Taq polymerase).
Free nucleotides.
Buffer solution.
Thermocycler to vary temperatures for the different stages.
What is the function of primers (forward and reverse)?
They are short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the DNA or RNA being copied.
They define the region that is to be amplified by identifying to the DNA polymerase where to begin building the new strands.
They also prevent the strands from joining up again.
Why is Taq polymerase used in PCR?
It is thermostable, doesn’t denature at high temperatures needed for the PCR to occur (95oC)
What are the 3 key stages of PCR?
Denaturation
Annealing
Extension/elongation
What happens at the denaturation stage of PCR?
Separates the DNA into 2 separate strands.
The hydrogen bonds between the 2 strands are broken.
(95oC)
What happens during the annealing stage of PCR?
Adding primers to the 2 separated DNA strands.
Temperature cooled to 55-60 oC to help the primers bind to the DNA.
The primers signal to a polymerase where to start synthesising new DNA.
What happens in the extension stage of PCR?
New DNA synthesised.
2 Taq polymerase molecule attached to the 2 primers on the 2 DNA strands and move along the strand.
As they move along they create new “complementary” DNA by adding nucelotides.
72oC
What does bioinformatics do?
It stores and organises large amounts of data.
Facilitates access to data on DNA and amino acid proteins and databases of metabolic pathways as well as fast retrieval and sharing of information as well as algorithms and stats tests.
What is the format of bioinformatics?
It is universal.
What does bioinformatics allow scientists to do in terms of disease and genetic disease? What does this then allow?
Identify the source of outbreaks and potentially vulnerable populations in outbreaks (can then be useful in developing vaccines).
Can be used after sequencing to identify alleles that cause genetic disease.
This then allows computer modelling of new protein structure from the base sequence.
What is the definition of computational biology?
The study of biology using computational techniques.
What is computational biology needed for?
Analysis of large amounts of data.
What does computational biology actually do?
It uses the data stored in bioinformatics to build theoretical models of biological systems which can be used to predict what will happen in different circumstances.
What does computational biology allow for?
Rapid processing of data.
Prediction of amino acid sequences.
Algorithms and stats tests.
EG analysing DNA sequencing to work out 3D structures of proteins and for understanding molecular pathways such as molecular pathways such as gene regulation.
How does computational biology and informatics link together?
Computational biology can use bioinformatics to make predictions about the structure and function of a synthetic protein using DNA sequences.
