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
Taqman Probes
- Increases the specificity of Q-PCR
- Have two primers and a probe specific to a gene. Probes sits between the primers and also bonds to the gene we are interested in
- Probe contains a fluorescent protein and a quencher
- Together on the probe, no fluorescence is given off as quencher quenches the fluorescence
- During a round of PCR, the primers and probe will hybridise to the specific region, and will get extension
- DNA becomes double stranded but doesn’t rely on it for the fluorescence to be given off
- As polymerase reads along, starts to degrade the probe
- The fluorescent proteins get separated from the quencher and fluorescence is given off
-Need to have lots of probe in reaction as each time its getting broken down.
Real time Results
Amplification plots are designed when the fluorescent signals from each sample is plotted against cycle number.
Gene expression
- All cells within a complex multicellular organism contain the same DNA but composed of different cell types
- Combination of genes that are expressed or repressed dictate cellular form and function
- Gene expression is regulated by signals from both within and outside cells and the interplay between these signals and the genome affect essentially all cellular proteases
Cell extrinsic regulation factors
=Environmental cues (outside the cell)
- Cues originate from other cells within the organism or from the organism’s environment
- Within the organism, cells communicate with each other by sending and receiving secreted proteins
- These signalling molecules trigger intercellular signalling cascade that ultimately cause changes in expression
Cell intrinsic regulation
=Intracellular cues (inside the cell)
- DNA can be modified which affects gene expression
- DNA and histone proteins can be chemically modified by cells machinery
- Chromatin modifications can affect gene expression by changing the accessibility of genes to transcription factors
- Chemical modifications: DNA methylation, histone modifications (methylation and/or acetylation)
Two major regulatory mechanisms
- Area around a prospective transcription zone need to be accessible
- Controlling the amount of gene product synthesised during the initiation of transcription.
Regulation at the structural level
- Eukaryotic DNA is packaged into chromatin
- Chromatin structure is directly related to the control of gene expression
- Chromatin structure begins with the organisation of DNA into nucleosomes
- Nucleosomes may block RNA Pol II from gaining access to promoters
Methylation
- Addition of -CH3 to DNA or histone proteins is associated with control of gene expression
- Clusters of methylated cytosine nucleotides bind to a protein that prevents activators from binding to DNA
- Methylated histone proteins are associated with inactive regions of chromatin
Acetylation
- Histones can be acetylated and deacetylated on lysine residues in the N terminal tail
- Acetylation removes the positive charge on the histone decreasing the interaction of the histone with the negatively charged phosphate groups of DNA
- Condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription
Regulation of Transcription
Controlling gene expression is often accomplished by controlling transcription initiation
Cis acting elements
- regulate the initiation of transcription.
- DNA sequences that serve as attachment sites for DNA binding proteins
Trans acting elements
-are regulatory proteins or miRNA that bind to cis acting elements. Activate or repress expression of the target gene
Cis acting elements
Specific DNA sequences which regulate transcription of one or more genes.
Comprises of two interacting parts:
Basal promoter elements
- binds accessory transcription initiation factors
- binds the important machinery to the RNA to be produced
Enhancer elements
-binds regulatory factors that help drive the expression of the RNA
Basal promoter
- Consist of a CAAT box, GC box, TATA box
- Ensures RNA pol II is in the right place and direction
TATA box
- required for RNA pol binding
- composed of TBP and TAFs
CAAT box
- required for genes to be transcribed in sufficient quantities
- absent from genes that encode proteins used in virtually all cells
GC box
-binding site for a protein called SpI (important transcription factor)
Enhancer
- DNA sequence which bind specific trans acting elements= activators
- These elements can interact directly or indirectly with basal factors at the promoter
- Can increase transcription 100-fold above basal levels
- Enhancers bind special transcription factor proteins that increase the rate of transcription
Silencer
- Negative regulation element
- Binds transcription regulation factors termed repressors
- Upon binding, RNA pol is prevented from initiating transcription thus decreasing or fully suppressing RNA synthesis.
Trans acting elements
Binds to gene promoters and enhancers. Different types of proteins bind to each other of the cis acting elements
- Basal factors bind to promoters: required for the binding of RNA polymerase to DNA
- Activators and repressors bind to enhancers: increase transcription in certain cells or in response to signals.
Post translation regulation
-Control of gene expression usually involves the control of transcription initiation
-But gene expression can be controlled after transcription with mechanisms such as:
RNA interference
Alternative splicing
RNA editing
mRNA degradation
RNAi
- Involves the use of small RNA molecules
- Enzyme Dicer chops dsRNA into small pieces of RNA
- miRNA bind to complementary RNA to prevent translation
- siRNAs degrade particular mRNAs before translation
Another level of regulation
Alternative splicing
- Introns are spliced out of pre-mRNAs to produce the mature mRNA that is translated
- Alternative splicing recognised different splice sites in different tissue types
- The mature mRNAs in each tissue posses different exons, resulting in different polypeptide products from the same gene
RNA editing
- RNA editing creates mature mRNA that are not truely encoded by the genome
- RNA editing is tissue specific
mRNA Degradation
- Mature mRNA molecules have various half life depending on gene and location expression
- The amount of polypeptide produced from a particular gene can be influenced by the half life of the mRNA molecule
