DNA Sequencing Flashcards
What does DNA sequencing do?
- Finds out order of nucleotides within a molecule.
What are the different types of sequencing?
- Sanger sequencing.
- High throughput sequencing.
- Bioinformatics.
What happens to the gene to be sequenced?
- Before PCR.
- Clone gene in vivo.
- Cut gene using restriction enzyme.
- Insert into a plasmid and insert into E. Coli.
- Let E. Coli divide.
- Extract plasmid and cut out restriction enzyme.
What happens during Sanger sequencing?
1- 4 reaction dishes.
2- DNA polymerase, 4 bases (A,T,C,G), primers added.
3- Add DNA that has been cut using restriction enzymes into a fragment.
4- Add modified bases which are radioactive - 1 per dish.
5- DNA polymerase adds bases onto the primer and produces many new strands.
6- ddNTP terminates chain -> fragment ends.
7- This produces many fragments which are all different lengths.
8- Separated during electrophoresis.
9- Nucleotide at end of each fragment read according to radioactive label.
What is throughput sequencing?
-1st automated DNA sequencing machine made in 1968.
- Used fluorescence hot auto radiography.
What happens during pyrosequencing - 1996?
1- Cut up DNA fragments mechanically using nebuliser.
2- Degraded into single strands and immobilised.
3- Primer added.
4– Incubated with enzymes: DNA polymerase, ATP sulphurylase, luciferase, apyrase.
5- Incubated with substrates: APs and luciferin.
6- Incubated with activated base ATP/ TTP/ CTP/ GTP
For pyrosequencing what is the amount of light generated proportional to?
To the amount of ATP and therefore nucleotides.
What is bioinformatics?
- Develops and uses computer software to analyse, store and organise biological data.
- DNA, RNA, protein.
- Data is universal and shared automatically.
E.g. sequencing data used to study genotype, phenotype correlations to treat disease.
What does comparative gene mapping do?
- Identifies importance of different genes.
- See tiny changes in genes cause different features.
- Comparing species identifies evolutionary relationships.
What does synthetic biology allow?
Knowing DNA sequence -> you can find out the amino acid sequence.
Designs and builds biological devices and systems.
E.g synthetic new proteins, new medicine.
What are the different uses of synthetic biology?
Building biological systems from artificially made molecules.
Redesign biological systems to perform better and include new molecules,
Designing new biological systems and molecules that don’t exist already.
What is pharmacogenetics?
Can figure out if a drug will work by testing someone’s genome.
Doctors can prescribe a drug that works rather than “one fits all”.
How does sequencing genome work?
1- mapped using microsatellites to identify genes.
2- Microsatelities are short pieces of repetitive sequences of 3-4 base pairs.
3- Samples of genome sheared into small sections.
4- Placed in bacterial artificial chromosomes and bacteria to make clone libraries.
