Steve's Flashcards
High throughput Sequencing
- Automated Dye terminator sequencing method used in the majority of sequencing
- Old methods are expensive and limited for large scale sequencing projects
- Allows thousands/million of sequences at once
- Intends to lower the cost of DNA sequencing and speed up the generation of genetic data.
4-5-4 Pyrosequencing
-Method amplifies DNA inside water droplets in an oil solution
-Each droplet contains a single DNA template attached to a single primer-coated bead that then forms a clonal colony
(Bead has multiple copies of the reaction)
-The sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzyme
-Uses luciferase to generate light for detection of the individual nucleotides added one base at a time to the DNA
(Since light is given off, easy to tell if nucleotides have been added)
Illumina (Solexa) Sequencing
- Technology based on reversible dye terminators
- DNA molecules have adaptor attached. Adaptors attach DNA to slides
- Amplification of DNA so local clonal colonies are formed
- 4 types of reversible terminator fluorescently labelled nucleotides are added and non incorporated nucleotides are washed away.
- Camera takes images of the fluorescently labelled nucleotides
- Dye are terminal 3’ blocker is chemically removed from the DNA allowing the next cycle
Amplify the signal so it can be read. Can add nucleotides at the same time as different colours are given off
Ion Torrent Sequencing
- Based on the detection of hydrogen ions that are released during the polymerisation of DNA
- Microwells containing a single template DNA strand is flooded with a single type of nucleotide
- If the nucleotide is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand.
- Each time H+ is given off, get a signal
Relies on the natural process of how nucleotides are added.
MinION
- USB powered DNA sequencer, laptop power
- Uses ‘disruptive nanopore based technology’
- Sequence DNA, RNA and proteins
- Nanopore is an organic molecule penetrated by a very small hole
- Nanopore is mounted on the membrane
- Voltage difference placed between the two holes of the fluid. Nanopore hole forms a path
- Fluid contains mobile ions. Ion current passes through the nanopore centre taking nearby molecules, proteins, RNA, DNA with it.
- Molecules disrupt the flow of ions in a characteristic manner which can be detected and interpreted.
Advances in sequencing techniques benefitting human genetics.
- Lower costs
- Easier to use
- Can use it in the field
- High throughput sequencing allows production of 1000s or millions of sequences at once
- Take longer reads
DNA sequencing
- Determines the order of nucleotide bases in DNA (A, G, C, T)
- Nucleotide order gives the amino acid order
- -Genomic structure and function
- -Cellular gene expression
- -Protein structure and function
- Allows us to determine a persons unique genetics. Look for differences between individuals.
DNA sequencing:
Chain termination sequencing
- Uses dideoxynucleotide phosphates (ddNTPs)
- Normal nucleotides have an -OH, ddNTPS have no modified OH group
DNA sequencing:
Sequencing reaction
- Polymerase adds complementary nucleotides to the growing chain until a ddNTP is incorporated randomly
- Terminates DNA strand extension, resulting in DNA fragments of varying lengths
DNA sequencing:
Sequencing Reaction Gel
- Different length strands can be lined up by size to determine DNA sequences
- Bands indicate DNA fragment after incorporation of ddNTP
- Position of the different band are then used to read the DNA sequence
DNA sequencing:
Gel Electrophoresis
- Each type of ddNTPs is fluorescently labelled with a different colour
- Laser reads the gel to determine the DNA sequence
RT-PCR/ q-PCR
- Amplify and simultaneously quantify target DNA
- Quantity can be either absolute number of copies or a relative amount when normalised to DNA input or additional normalising gnees
Two methods for detection of products in RT-PCR
- Dye fluorescence
- Non specific fluorescent dyes that intercalate with any double stranded DNA - Fluorescent Reporter Probes
- Sequence specific DNA probes labelled with a fluorescent reporter
- Detection after hybridisation of the probe with its complementary DNA target.
Dye Fluorescence
e.g. SYBR Green
- DNA binding dye binds to all dsDNA in PCR, causes fluorescence of dye
- Increase in DNA product during the PCR, increases fluorescence intensity
- PCR reaction is prepared as usual with the addition of fluorescent dsDNA and run in a Q-PCR machine
- After each cycle, the level of fluorescence is measured with a detector
- Dye only fluoresces when bound to the dsDNA (PCR product)
- Allows DNA concentration to be quantified.
Dye fluorescence problems
- dsDNA dyes will bond to all dsDNA PCR products e.g. primer dimers (primers binding to themselves)
- Potentially interfere with or prevent accurate quantification of the intended target sequence
