12 - Mutation Detection 2 Flashcards
Sanger sequencing
ssDNA used as template in PCR with single extension primer, DNA polymerase, dNTPs and ddNTPS which terminate elongation.
Maxam-Gilbert cleavage sequencing
DNA molecules are subject to chemical treatment to partially modify specific bases and then cleavage to generate fragments
of varying size –> size resolved by gel electrophoresis to determine sequence
Automated Sanger sequencing
- PCR with fluorescent, chain terminating ddNTPs
- Size separation by capillary gel electrophoresis
- Laser exciton & detection by sequencing machine
Illumina DNA sequencing
- Slide flooded with fluorescently labelled nucleotides and DNA polymerase
- One base added at time
- Image taken of slide showing base that has been added
- Computer detects the base at each site based on colour
- All of the sequence reads will be the same length, as the read length depends on the number of cycles carried out.
examples of optical devices used in characterisation of DNA
- Periscopes, microscopes, telescopes and cameras
- enabled observed chromosome doubling, chromosome theory and jumping gene
Circulating free DNA sequencing
Circulating free DNA (cfDNA) are degraded DNA fragments released to the blood plasma. cfDNA can be used to describe various forms of DNA freely circulating in the bloodstream, Including circulating tumour DNA (ctDNA), cell-free
mitochondrial DNA (ccf mtDNA), and cell-free fetal DNA (cffDNA).
Applications of cfDNA sequencing
- Pre-natal screening of foetal DNA in mum’s plasma
- Screening plasma for cancer-associated mutations
Steps of Next Generation Sequencing
- Library preparation
- Cluster growth
- Sequencing
- Imaging
How do ddNTPS stop sequencing
Lack the 3’-OH group required for phosphodiester bond formation, therefore replication cannot occur (3’- H instead)