what is DNA sequencing
working out the sequence/order of nucleotide bases
what is meant by the term genome
the entire genetic material of an organism
why is knowledge of DNA sequences useful
understand evolutionary relationships, comparing genomes between individuals and species and to be able to predict amino acid sequences
what is the chain termination method - manual sanger sequencing
- uses modified nucleotides called dideoxynucleotides (terminator nucleotides)
- these pair with nucleotides that have a complimentary base
- when DNA polymerase encounters a dideoxynucleotide on the developing strand it stops replicating
what do dideoxynucleotides do
stop replication as it cannot form a phosphodiester bond between it and a nucleotide
what is different between the structure of deoxynucleotides compared to terminator nucleotides
- have less hydroxyl groups
- have no condensation reaction
what is the process of manual singer sequencing
1- many copies of a single stranded piece of DNA are used as templates to sequence
2- a primer is added so that DNA polymerase can bind and begin copying the DNA strands
3- the DNA and primers are added into 4 identical test tubes with free normal nucleotides, for DNA polymerase to add to the growing chain a small amount of terminator nucleotides are also added randomly
4- in each tube many copies of the DNA molecule are made but all different lengths
5- the 4 tubes are then loaded into agar jelly to undergo electrophoresis
6- by reading the pattern scientists can work out the order of bases in the DNA sequence
what is the process of automated sanger sequencing
1- each type of terminator nucleotide is labelled using a specific fluorescent dye
2- the single stranded DNA chains are separated according to mass using capillary electrophoresis (works same as electrophoresis but is just in capillary minute tubes)
3- a laser beam is used to illuminate all of the terminator nucleotides and a detector then reads the colour and position of each fluorescence
4-the detector feeds the information into a computer where it is stored or printed out for analysis
what are the uses and strengths of sanger sequencing
- gives high quality sequence for relatively long stretches of DNA (up to 900 base pairs)
- it is typically used to sequence individual pieces of DNA such as bacterial plasmids or DNA copied in PCR
what are limitations of sanger sequencing
- expensive and inefficient for larger scale projects e.g. sequencing of an entire genome
what is next-generation sequencing
- any method of DNA sequencing that has replaced the sanger method
- a set of massively parallel sequencing technologies that allow for the simultaneous sequencing of many DNA fragments in a high throughput manner.
what is next generation sequencing also known as
high-throughput sequencing
what is different about NGS and the sanger method
- NGS can generate millions or billions of short DNA sequences in a single sequencing run where as the sanger method which sequences one fragment at a time
NGS in comparison to sanger method is …
- highly parallel and fast as many sequencing reactions take place at the same time
- micro scale as reactions are tiny and many can be done at once on a chip
- low cost as it is much cheaper
- shorter length as it reads typically range from 50-700 nucleotides in length
what is the system of pyrosequencing
1- a piece of DNA is cut up into fragments (400-600bp) using restriction enzymes
2- they are made single stranded and are the template DNAs, they are immobilised on a bead
3- a primer is added and DNA incubated with DNA polymerase and other enzymes
4- then one of the 4 specially activated nucleotides are added at any one time
5- if the next nucleotide is attached, there is a reaction and light is emitted which can be detected by a camera, if 2 of the same nucleotide are next to each other double the light is emitted
6- any unused nucleotides are degraded before the next base is added
what is nanopore sequencing
- currently being developed and invloves single strands of DNA are pulled through a pore (as each each base is pulled through the pore its conductivity is measured). Any length of DNA can be sequenced
what are uses of DNA sequencing
- human genome project
- comparing the genome of individuals
- comparisons between species
- evolutionary relationships
- epidemiology
what are the 3 stages of PCR
- denaturation
- annealing
- extension
what does denaturation involve
- heating the section of DNA to 94-96°c through the use of a thermal cycler
- this breaks the hydrogen bonds between the 2 strands
- leads to single stranded DNA
what does annealing involve
- cool both strands to 54-60°c
- and primers added
- the cooling allows the primers to bind to the strands
what are primers
- short strands of DNA that are complimentary to the bases at the start of the target fragment
- tell DNA polymerase where to attach
why is taq polymerase used
- doesn’t denature at high temperatures
- many cycles of PCR can be carried out using the same enzyme
what does extension involve
-heated to 72 and taq polymerase attaches
-72° is the optimum temperature
- new strands are formed and the target DNA is doubled
what is the process of electrophoresis
- DNA fragments are loaded into wells
- an electric current is passed through the electrophoresis tank which pulls the DNA fragments through the agar jelly towards the anode as DNA is negatively charged
- this separates the DNA fragments into size
- DNA fragments of the same length form a band that can be seen by the eye if it has been stained with a DNA-binding dye
