DNA sequencing Flashcards
What is DNA sequencing?
The process of determining the precise order of nucleotides within a DNA molecule
What is Dideoxy chain termination (Sanger sequencing)?
DNA sequencing technique:
- low error rate and highly reliable
- ‘gold standard’ technique meaning other sequencing techniques are compared to it
What is Automated DNA Sequencing by ABI 3730?
Can perform sequencing of multiple samples simultaneously, with a very high accuracy
What is the disadvantage of automated DNA sequencing?
only performs the separation of labelled DNA and determined the sequences, however the whole sequencing process still requires considerable hands preparation prior to sequencing
Describe the Basic Dideoxy chain termination steps.
1) Single stranded DNA template generated
- done by DNA denaturation
2) Enzymatic Sequencing Reaction
- DNA polymerase makes multiple copies of the DNA
3) Size Separation of Products by Electrophoresis
- labelled DNA molecules separated by size
4) Detection of Reaction Products
- sequential detection of the terminating nucleotide to identify base
5) Readout of Sequence
- reconstructing the sequence
What are the Requirements of Dideoxy chain termiantion?
4 reaction mixtures set up including:
Primer needed to anneal/hybridise to template strand
DNA Polymerase recognises this partially dsDNA molecule and forms initiation complex
Elongates this using dNTPs from the 3’OH terminus of the primer in a 5’ to 3’ direction. Ester bond formed between free 3’OH group and phosphate on dNTP. Mg2+ required as a cofactor.
One kind of ddNTP (labelled with different fluorescent molecule) added to each of the 4 reaction mixtures to terminate the reaction, at a low molar ratio compared to the dNTPs. If a ddNTP is incorporated into the strand during elongation, elongation is terminated because there is no free 3’OH group on ddNTP to react with phosphate of dNTP, and this prevents further extension. Polymerase then dissociates and strand is released from enzyme.
Describe the process of size seperation.
Size separation of each of the molecules produced in the 4 different reaction mixtures can be done by gel electrophoresis:
-larger molecules are retarded (delayed) to a greater extent as as a consequence move through the gel matrix from the negative electrode to the positive electrode more slowly
> fastest moving molecules are the smallest molecules, meaning they were the terminating nucleotides closest to the primers and to the 5’ end of the elongating strand
Describe the process of detection of reaction products.
Detector shines light onto molecules which will fluoresce at specific wavelength
By capturing the fluorescence of these molecules, you can determine which specific population of terminal nucleotides is present within the molecules
Electropherogram is produced where fluorescent measurements generate a trace and “base calling” is automated. A graph is produced where individual bases are represented by each individual coloured trace. The software then reconstitutes from that trace and determined what the actual sequence represented by that electropherogram is.
What is the Difference between dideoxy sequencing and PCR?
both very similar as they both use DNA polymerase, but:
- dideoxy sequencing only uses a forward primer
- amplification is not exponential in dideoxy sequencing
- acidification of the reaction as the formation of the ester bond during elongation produces pyrophosphates and H+ ions
Give the Uses of Dideoxy Chain Termination.
HEALTH
-confirmatory test for specific gene mutations (except low frequency mosaicism) in patients with suspected genetic diseases
RESEARCH
