Lecture notes
Advanced Molecular Biology lecture notes
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- University Of Westminster (UOW)
Lecture notes on the basic Advanced Molecular Biology course. Simple and easy to understand and learn for an exam. Has the basics.
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Adv. Molecular BiologySanger Sequencing VS NGS
Sanger Illumina Nanopore
Mixture of fluorescent Mix of fluorescently labelled No DNA synthesis involved,
ddNTPs to terminate the reversible chain terminators DNA chains are read directly
chain
1000 base pairs read length Short reads (e.g. ~70 bp) but Very long reads possible into
maximum a lot of them the millions of base pairs
Low amount of DNA Very high producKon of DNA High producKon of DNA
sequence produced sequence sequence
(hundreds of machines
running for months to
sequence a genome)
Low error rate High error rate High error rate
Sanger Sequencing Steps
- Pure sample e.g. PCR product or plasmid
- Add one sequencing primer, DNA polymerase, dNTP mix, fluorescent ddNTP mix and
use a thermocycler to run the sequencing reacKon
- Clean up the reacKon (take out the excess ddNTPs)
- Run on a capillary electrophoresis
- Computer calls the sequence
- SS reads for maximum 1000 bp
Sanger Sequencing Principles
- DNA denaturaKon: DNA double stranded template is denatured to single stranded
DNA fragments
- Primer annealing: sequencing primer anneals to the template DNA strand giving a 3’
OH group for DNA polymerase
- DNA synthesis: DNA polymerase extends the primer by incorporaKng regular dNTPs
and a labelled ddNTP
- Gel electrophoresis: ResulKng DNA fragments of varying lengths are separated on a
polyacrylamide gel based on size
- DetecKon of fluorescent signals: Each labelled ddNTP is mixed, a fluorescent signal is
shown and the DNA bands are visualised.
PCR vs Sanger Sequencing
PCR Sanger Sequencing
dNTps dNTPs and ddNTPs
Two primers that copy both strands One primer only copying one strand
Amplify one sequence from a mixture Uses a fairly pure and concentrated
template such as a PCR product or a
template
Pa@ern of inheritance mDNA
- Mitochondria are inherited from the mother, mitochondria in the sperm are
degraded aYer ferKlisaKon
- GeneKc diseases involving mutaKon in the mitochondrial DNA are passed on
exclusively by the mother
- Offspring of affected mothers are affected but none og the offspring of the affected
fathers would be
, - Muscle weakness is one of the phenotypes seen with mutaKons in mtDNA genes
because of the defects in mitochondrial funcKon
Mitochondrial disorders
- MutaKon in mtDNA causes mitochondrial disorders
- Single nucleoKde variaKons – involves 1 base
- Length variaKons – stetch of mtDNA has a variable length of stretch of bases (C)
- DeleKons/inserKons – loss or gain of regions of mtDNA
- Disorders because of inherited geneKc mutaKons in mtDNA
- Hereditary mitochondrial disorders are inherited by the mother
- Disease phenotypes vary because of heteroplasmy
- Heteroplasmy is the presence of mulKple organellar genomes within a cell or
individual (same genome)
- VariaKons in heteroplasmy arises due to random segregaKon of mitochondria during
mitosis or meiosis
- Extent of heteroplasmy can affect disease phenotype – cells with more mitochondria
with variant sequences has a greater likelihood of manifesKng disease phenotype
- Prevalence: 1:5000 and affects all age groups
- Pathophysiology: bioenergeKc failure, lacKc acidosis, oxidarice stress
- MulK-systemic – however, most affected cell types are those which are heavily
dependent on energy, central nervous system, heart and skeletal muscle, liver
Species idenDficaDon using mtDNA
- Wild-life poaching
- PreservaKon of endangered species
- Food contaminaKon
- Criminal cases to work out if samples at the scene of the crime are human or animal
- Animal a`acks
- Advantages: species can be idenKfied by rouKne molecular biology methods rather
than needing a skilled and specialist taconomist
- Sequencing a region of the mitochondrial genes cytochrome c oxidase subunit 1 or
cytochrome b is sufficient to idenKfy animal species
- 2000 mitochondria per mammalian cell so much easier to amplify than nuclear DNA
hence less sample is needed.
Mitochondrial haplogroups
- Disease causing mutaKons in mtDNA, hundreds of addiKonal ancient mutaKons
which have developed over millions of years of human evoluKon
- Mitochondrial haplogroups are combinaKon of polymorphic markers
- Due to maternal inheritance, haplogroups are conserved across generaKons
- Different mutaKons can serve as markers of ancestral origin
- PhylogeneKcs use present-day informaKon such as sequence data to infer
evoluKonary history
mtDNA in forensic analysis
- Amount of extracted DNA is small or suscepKble to degradaKon (hair, bone, teeth)
- High copy number, as there are thousands of copies of mtDNA per cell
- RelaKvely small size of genome means high likelihood of finding intacts porKon of
mtDNA that can be amplified and sequences
- No recombinaKon of the mitochondria it may have less ability to idenKfy individuals
than analysis of nuclear DNA, so mainly useful when DNA is limited
, Outline design of an experiment Sanger Sequencing
- Before sanger sequencing, make sure there is a pure DNA sample, PCR is done to
amplify the gene of interest
- Sanger sequencing then happens
- IsolaKon of DNA
- PCR to amplify the gene (Design primers, assay uses temple, two primers, dNTPs,
DNA polymerase and thermocycler)
- SeparaKon of the sequencing products using capillary electrophoresis or gel
electrophoresis
- Calling the sequence from the sequence trace and comparing with individual to
decide if the paKent has a mutaKon in the gene
How chemistry of sanger sequencing enables the producDon of the sequencing trace
- DNA polymerase can add either a dNTP or a ddNTYP (dedioxynucleoKde phosphate)
to a DNA chain
- AddiKon of a ddNTP prevents further extension of the chain (terminates it)
- Sanger sequencing uses a mix of dNTPs and ddNTPs, so a ladder of different length
products is produced each one base longer than the previous
- ddNTPs are labelled with specific fluorescent dyes, their fluorescence is read to
produce the sequencing trace
Sanger Sequencing VS Next generaDon sequencing
Sanger sequencing NGS
Chain terminaKon method Parallel sequencing of mulKple DNA
fragments
Shorter bases 1000 Longer reads
Lower throughput Higher throughput
Expensive Cheaper
Time consuming Fast turnaround Kme
Limited automaKon High degree of automaKon
Low error rate High error rate
Single gene analysis More complex data analysis
Needs larger dna Small amount of dna
ddNTPs Illumine
Principle of PCR
DenaturaKon – 98C – double stranded DNA template is heated to separate strands
Annealing – 65C – primer binds to their complementary seaeunces on the DNA
ElongaKon – 72C – DNA polymerase synthesises new DNA strands using primer
Cycling – Repeated – 3 steps are done 20-4- Kmes to amplify DNA
AYer PCR – amplified DNA can be used in various other applicaKons
Sanger Sequencing Principles
DNA denaturing – DNA double stranded template is denatured to get single stranded DNA
fragments
Primer annealing – sequencing primer anneals to the template DNA strand giving 3’ OH
groups for DNA polymerase
DNA synthesis - DNA polymerase extends the primer by incorporaKng regular dNTPs and a
labelled ddNTP
Gel electrophoresis – resulKng DNA fragments of varying length are separated on a
polyacrylamide gel based on size
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