DNA Sequencing Flashcards
What is another name for Sanger Sequencing?
Dideoxy chain termination
Why is Sanger sequencing so reliable?
Very robust – with low error rate
Hence it is a “gold standard” technique - used as a comparative for other techniques
Who discovered Sanger sequencing?
Method developed by Nobel Prize Winner Fred Sanger to sequence DNA in 1977
Technology has improved, the technique modified and semi-automated
How are samples prepped for use by the ABI 3730?
samples prepared by dideoxy chain termination on a large scale by robotics
How many samples are processed by ABI 3730?
Reads length of up to 900bp & 99.95% accuracy
Handles 48 or 96 Samples simultaneously
>1000 samples per day
How does DNA separation occur via the ABI 3730?
But only performs the separation of labelled DNA & determines the sequence – requires considerable hands on manipulation
What role did the ABI 3730 play in sequencing human genome?
Technique was used to sequence the Human Genome
Produced 23 giga bases of sequence
But took 13 years & $2.7 bn to complete
Outline the Dideoxy chain termination steps
- Template used in a sequencing reaction using DNA
Polymerase to produce multiple copies of the
complementary strand - Products separated based on size using capillary
electrophoresis - Sequential detection of terminating nucleotide to
identify the base - Readout of sequence reconstructed into base sequence
What template is used in Dideoxy chain termination?
Template : either a clone (e.g. plasmid) or a PCR product Required in:
- High degree of purity
- Large amounts
How are products detected in dideoxychain termination?
Individual molecules are terminated by a particular dideoxynucleotide determined by the sequence, so the original sequence can be detected and reconstructed from the readout
How is dideoxy chain termination similar to PCR?
Some protocols cycle through repeated temperatures
It also uses a DNA polymerase
How does PCR and dideoxy chain termination differ?
Dideoxy chain amplification is limited and NOT exponential
Dideoxy chain termination uses a single forward primer
When is DNA polymerase used in Sanger sequencing?
If cycling is performed a thermostable polymerase so would be necessary and is usually used
Outline how sequencing is carried out using DNA Polymerase
DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides
- A single stranded oligonucleotide (primer) is bound to
template - Polymerase recognizes DNA structure, forming an
initiation complex - Commences elongation from 3’ terminus of primer in a
5’ to 3 ‘ direction
What are the requirements of DNA Dependent DNA polymerase?
Requires: - Template strand that extends beyond a primer - Free 3’ OH group on primer - All 4 Deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP) - Mg2+ ions
Describe the structure of the elongating DNA Strand
5’ end - 5’ Carbon is attached to triphosphate
Phosphodiester bond forms backbone of DNA molecule
Base has a hydroxyl group at 3’C position allowing the formation of another phosphodiester bond with the added base
What are the byproducts of the elongation strand?
=> terminal phosphates are released producing inorganic pyrophosphate
=> hydrogen released forming H+ ions
=> during PCR release of H+ causes acidification of the reaction
Why does acidification not occur during Sanger sequencing?
During Sanger sequencing a relatively low number of H+ ions are released ∴ acidification doesn’t occur, whereas PCR is exponential so produces a lot more H+
What are the requirements of dideoxy chain sequencing?
- Template strand that extends a primer forming a partial duplex - Free 3’ OH group on the primer - All 4 Deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP) - All 4 Dideoxy nucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP) - Mg2+ ions`
How is DNA elongation terminated?
DNA elongation is terminated by addition of dideoxynucleotide
How does the addition of a dideoxynucleotide terminate elongation?
No OH’ on 3’C on ribose ring so elongation can’t occur anymore once dideoxynucleotide has been added
What feature enables dideoxynucleotides to be identified?
Each dideoxynucleotide suppresses a different wavelength and so can be detected by fluorescent markers
How does sequencing occur in the presence of dideoxynucleotides?
DNA is mixed with the reaction components including both dideoxy and deoxy-nucleotides
- Polymerase commences elongation from the 3’ terminus
- As the enzyme encounters a particular nucleotide in the
sequence it picks out and incorporates a
complementary nucleotide into the elongating strand. - If a dideoxy nucleotide is incorporated into the strand,
then elongation is terminated otherwise elongation
continues
How big are the reaction products produced via Sanger sequencing?
The reaction products vary in length terminated by ddNTP
