DNA Sequencing by Dideoxy-chain termination Flashcards
What is another name for DNA sequencing via dideoxy-chain termination?
Sanger sequencing
What is the read length and accuracy of the modern method of dideoxy chain termination?
- Read length - up to 900 bp
- Accuracy - 99.5%
What does that fact that the machines used for dideoxy-chain termination can only perform the separation of labelled DNA & determine the sequence mean for the ease of the technique overall?
It means that the technique is quite time-consuming as considerable hands on preparation, e.g. adding the reagents, needs to be done before sequencing can occur which is a drawback
What are some of the similarities between Dideoxy-chain termination and PCR?
- Both use a DNA-dependent DNA polymerase
- Can also go through multiple cycles of heating and cooling like PCR if you want to amplify the template
Dideoxy chain termination only uses a single forward primer. What does this mean if you go through multiple cycles of it like you would in PCR?
- It means that amplification of the template will be limited and not exponential like it is in PCR
Give a brief overview of the different steps involved in sequencing by dideoxy chain termination
- Template goes through enzymatic sequencing reaction
- Size Separation of products produced by the enzymatic sequencing reaction by capillary electrophoresis
- Detection of reaction products using labelled terminating nucleotide
- Use the readout of the sequence to re-construct the original template sequence
Describe the first steps involved in the sequencing reaction performed as part of dideoxy chain termination
- A clonal population of DNA template molecules are placed in the reaction mixture along with an oligonucleotide primer, DNA-dependent DNA polymerase, deoxy and dideoxyribonucleoside triphosphates and a buffer
- The reaction mixture is then heated up so the DNA template molcules denature to form single stranded DNA template molecules
- The oligonucleotide primer anneals (binds) to the single stranded template forming partial duplexes.
- This allows the DNA-dependent DNA polymerase to recognise and bind to these partial duplexes to form initiation complexes
Describe the elongation step involved in the sequencing reaction performed as part of dideoxy chain termination
- Once the DNA-dependent DNA polymerase forms an initiation complex with the partial duplex it then incorporates a deoxyribonculeoside triphosphate (dNTP) into the elongated strand
- Once incorporated the DNA-dependent DNA polymerase facilitates the reaction between the OH group on carbon 3 of the pentose sugar of the previous dNTP and the phosphate group on carbon 5 of the pentose sugar of the incorporated dNTP
- This reaction produces an ester bond between the 2 nucleotides and causes the elongation of the elongating strand by one nucleotide
- The reaction also produces a H+ ion, and pyrophosphate
Describe the termination step involved in the sequencing reaction performed as part of dideoxy chain termination
- Within the reaction mixture there as well as dNTPs there are fluorescently labelled dideoxyribonuleoside triphosphates (ddNTPs)
- Eventaully the DNA-dependent DNA polymerase will incorporate an ddNTP into the elongating strand at random
- When this happens elongation is terminated as the DNA-dependent DNA polymerase isn’t able to incorporate another dNTP or ddNTP into the elongating strand
Why does the incorporation of a ddNTP into the longating strand cause elongation to stop?
- Because ddNTPs lack an OH group on carbon 3 of the pentose sugar
- Without that OH group the DNA-dependent DNA polymerase isn’t able to facilitate the formation of an ester bond between that carbon 3 OH group and the phosphate group on carbon 5 of the pentose sugar of the next dNTP
- This means that no further nucleotides are able to be added to the elongated strand thus causing the termination of elongation
What do you end up with once termination of elongation occurs during the sequencing reaction
- You end up with a number of DNA molecules of different lengths with a particular ddNTP at the end of the elongated strand
Why are the reaction products of the sequence reaction sorted by size?
- Allows you to determine the sequence of the elongated strand
- This is done by identifying the terminal ddTNP found at the end of each sequence within the reaction mixture at the end of the reaction
How are the reaction products of the sequence reaction sorted by size?
- They are separated by gel or capillary electrophoresis
- The nucleic acid passes through a gel matrix by applying a voltage across two electrodes
- Negatively charged nucleic acid migrates towards the positive electrode.
- The matrix retards the molecules according to their size so those molecules that are larger are retarded to a greater extent and as a consequence move through the matrix more slowly
Why is a detector placed at the end of the capillary/gel matrix?
- The detector shines light onto the different populations of DNA found on the matrix
- Because each population represents a strand in which a ddNTP is incorporated at a different point within the elongated strand each population will fluoresce at a particular wavelength
- The wavelength that each population fluoreseces at will be used to identify which of the 4 ddNTPs was incoporated at the terminal end of that length of elongated strand
How is the fluorsecence pattern of each of the populations of DNA on the matrix used to determine the sequence of the elongated strand?
- Fluorsecence pattern used to determine which of the ddNTPs is incorporated at the end of the elongated strand
- Population of DNA molcules at the end of the gel/capillary matrix represent the elongating strands in which the ddNTP was incoporated closest to the oligonucleotide primer
- Because they were incorporated at the 3’ end of the primer it means they are closer to the 5’ end of the elongating strand.
- This means as you identify the ddNTP that was incorporated for each population from the end of the matrix to the beginning you can work out the sequence of the elongating strand from the 5’ end to the 3’ end
