Define DNA sequencing** & what is needed for **Sanger Sequencing?
DNA Sequencing: Process of determining order of nucleotide bases in a DNA molecule
For Sanger Sequencing…
- Terminator bases (A, T, C, G), which stop DNA synthesis when they complementarily base pair
- They have fluorescent tags to allow them to show up
- A primer - short sequence of nucleotides that joins to the 5’ end of DNA to allow extension of DNA
- A mix of normal nucleotides (A, T, C, G)
- DNA you want to sequence
- DNA polymerase
What is the process of Sanger Sequencing?
- DNA for sequencing mixed with a primer, DNA polymerase and an excess of normal nucleotides
- Mixer placed in a thermal cycler for PCR
- At 60oC the DNA polymerase builds new DNA strands by adding nucleotides as a complementary strand to the single strands
- Each time a terminator base pairs the DNA synthesis is terminated at that length, since the pairing of bases is completely random you get a mix of different lengths of DNA fragments
- This process is run in several cycles in the cycler, so you end up with DNA fragments terminated at every possible base along the whole sequence
- The DNA fragments are separated according to length by capillary sequencing/gel electrophoresis
- Fluorescent markers on the terminator bases identify each final base on a fragment, lasers detect the colours to create the order of the sequence
What advancements in sequencing techniques have there been?
High Throughput Sequencing
- Initially, using Sanger Sequencing was time-consuming and difficult
- New technologies have been developed to automate the process to speed it up
- Instead of gel or capillaries, sequencing takes place on a flow cells (a slide)
- Millions of DNA fragments are attached to a slide and amplified/replicated using PCR, forming clusters of identical DNA fragments
- The same coloured terminator base technique for sequencing is used
- It is known as ‘massively-parallel sequencing’ because the DNA fragment clusters are sequenced at the same time
Define:
Bioinformatics
Computational Biology
Bioinformatics: Development of software and computing tools to organise and analyse raw biological data
Computational Biology: Uses the organised and analysed data from bioinformatics to create theoretical models used to predict outcomes under different circumstances
What is genomics, and what is the human genome project?
Genomics: Field of genetics where DNA sequencing and computational biology analyses the structure and function of genomes
The human genome project analysed the sequence of thousands of human genomes
Computer analysis can compare genomes of individuals, revealing patterns in DNA that we inherit and diseases we are vulnerable to
What does sequencing genomes of pathogens tell us?
- Doctors to find the source of an infection – e.g. bird flu/MRSA in hospitals
- Doctors can identify antibiotic-resistant strains of bacteria, so they choose the correct antibiotics
- Culturing bacteria takes time
- Scientists can track the incidence of disease in epidemics
- Scientists can identify regions in a pathogen genome making them a target for the development of new drugs AND to identify genetic markers for vaccines
How can gene sequencing help in searching for evolutionary relationships?
- DNA sequencing has allowed organisms to be compared as basic mutation rate can be calculated to work out how long ago 2 species diverged from a common ancestor
- The more similar a DNA sequence, the closer they are related
- Scientists can build evolutionary trees
What is proteomics and how has gene sequencing helped it?
Proteomics: Study of amino acid sequencing of an organism’s entire protein complement
- There is a complex relationship between genotype and phenotype, there are more proteins than genes
- DNA sequence of a genome allows you to predict amino acid sequences and protein sequences
- However, not all genes code for the same amino acids you expect, some genes code for several proteins
What is synthetic biology?
Synthetic Biology: Designing and building biological devices and systems, to fulfil requirements such as: Providing food, diagnosing illness, producing medication
What are techniques used in synthetic biology?
- Genetic Engineering
- Use of biological systems in industry, e.g. using enzymes and the production of drugs from microorganisms
- Synthesis of new genes, replacing faulty genes, e.g. to treat cystic fibrosis
- Synthesising new organisms, e.g. new bacterium with different genomes
