DNA sequencing Flashcards
What is DNA sequencing
the process of determining the precise order of nucleotides within a DNA molecule
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
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
Basic Dideoxy chain termination steps
A multi stage process
- Single stranded DNA template generated done by PCR
- done by DNA denaturation - Enzymatic Sequencing Reaction
- DNA dependant DNA polymerase makes multiple copies of the complementary strand of the DNA template - Size Separation of Products (molecules) by Capillary Electrophoresis giving high resolution separation of molecules that differ in size by 1 single base
- labelled DNA molecules separated by size - Detection of Reaction Products
- sequential detection of the terminating nucleotide to identify base - Readout of Sequence
Requirements of Dideoxy chain termination
DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides
1. Single stranded oligonucleotide (primer) is bound to the template (anneal/hybridise to template) strand forming a partially double stranded structure (annealing is driven by the molar excess of the primer as it is in competition with renaturation - just like in PCR)
- The polymerase recognizes the dsDNA structure, then forms an initiation complex
- Elongates (extension) this using dNTPs from the 3’OH terminus of the primer. Mg2+ required as a cofactor.
- Phosphodiester bond formed between free 3’OH group and phosphate on dNTP
The reaction releases H+ ions and eventually acidifies it. Mg2+ required as a cofactor. - Chain termination: allows us to radilly hold the elongation
ddNTP does not have OH - cause termination of elongation, prevents further extension of the strand.
The dideoxy sequencing reaction is similar to PCR but NOT PCR
How?
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
What does DNA dependant DNA polymerase do?
Single stranded oligonucleotide (primer) is bound to the template and the polymerase recognizes the DNA structure, then forms an initiation complex
What are the 4 parts of the dideoxy chain termination reaction
Strand separation
Annealing primer
Extension
Chan termination
What does DNA dependant DNA polymerase require?
- A template strand that extends beyond a primer
- Free 3’OH group on the primer
- All 4 deoxynucleotide triphosphates
- Mg2+ ions as co factor and we need a buffer
difference between deoxy nucleotide triphosphate (dNTP) and dideocy nucleotide triphosphate (ddNTP) ?
ddNTP terminates elongation as it doesn’t have OH so cant continue
Why do the strands vary in length?
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.
However, where ddCTP is incorporated, elongation continues - so they all terminate at diff lengths
What is size seperation?
molecules vary in length and are terminated by ddNTP so size separation (ordering by size) allows us to determine the sequence of the new strand (due to their fluorescence) 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
Detection of Reaction Product
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.
Uses of Dideoxy Chain Termination
In health, to test for specific gene mutations and to confirm all types of mutation (nonsense, misense etc)
