Profiling and Sequencing Flashcards
What are the steps in DNA profiling?
- Purify the sample and extract DNA - extract from cells and separates from cellular components
- Quantify the amount fo DNA to make sure there is enough
- Use PCR to make copies of DNA
- STR analysis of DNA fragments - use gel electrophoresis to separate them
What is profiling?
- Looks at allele-level differences
- Provides information about familial relationships
- Useful in forensic science
- Relies on polymerase reactions
What is sequencing?
- Looks at single base-level sequence
- Provides information about proteins expressed
- Useful for biomedical analysis
- Relies on polymerase reactions
What are dNTPs?
Deoxynucleoside 5’-triphosphates
* Building blocks of new DNA
* Has an extra OH group which allows bridging to the next base pair on the phosphate
* Build off the 3’ end of the DNA
What do ddNTPs do?
Terminates DNA synthesis
* doesnt have the OH group on the 3’
What does the dNTPs do?
DNA synethesis monomers
What is Sanger Sequencing?
- Primer gets added
- DNA polymerase adds on dNTPs to replicate the DNA sequence
- Will then add a ddNTP which will stop polymerisation
- Will continue down the strand but the dNTPs wont bind to the ddNTPs
- Can then measure the length of this using gel and identify the specific ddNTP that is stopping polymerisation
Reading a PAGE gel and Sanger Sequence
- Position of bands tells you which positions have the ddNTP, since the final base will be that same ddNTP
- Then can test the sample 3 more times with the other ddNTPs and figure out the entire template sequence
What are teh 4 dNTPs?
- dATP
- dCTP
- dGTP
- dTTP
What are the four ddNTPs?
- ddATP
- ddTTP
- ddCTP
- ddGTP
Visualising a Sanger Sequence
- Label each ddNTP with a different coloured fluorescent dye
- run one PCR & one lane in a gel
- Place gel under UV light to visalise the different colours
- or use automated capillary electrophoresis and measure many samples at once
What is Next Generation Sequencing?
- Fragment genomic DNA using ultrasound to vibrate it
- Isolate individual fragments on the surface of a bead and amplify it
- Colour change electrically
- Add one base at a time & record which one is being added to determine the sequence of fragmetns
- Then use data reconstruction to detemine the full sequence
Sanger Sequencing vs NGS
Sanger Sequencing:
* Near perfect fidelity
* One read per run
* Up to 900 bases that can be read
* ~400 hours to read a million bases
* $2400 cost per million bases
NGS:
* Excellent if multiple reads combined
* Up to 3 billion reads per run
* Up to 15,000 bases that can be read
* 0.1 s to read a million bases
* $0.10 cost per million bases
Nanopore sequencing
- Portable
- Simple
- Potential applied
- Enzyme unwinds the double helix & passes a strand through a nanopore
- The channel in the nanopore is just the right size for the DNA strand to pass through
- Each DNA basr is a different size, so block nanopore differently - measured by change in currnet
What are the steps involved in DNA extraction?
- Lysis - breaks open the cell and nucleus to free DNA
- Precipitation - new sodium ions neutralise the negatively charged DNA and then alcohol is added to precipitate the DNA (form a solid)
- Purification - DNA precipitate is washed with alcohol to remove impurities, usually dissolved in water again
