TD: Nucleic acid detection + sequence determination Flashcards
What is the genetic code?
- The set of biological rules that dictate the translation of genetic information contained within the DNA into specific amino acids, and ultimately a specific protein
- Thus, a protein drug target is the product of a particular gene
Why is understanding of DNA good in terms of therapeuticd?
- Understanding of DNA will allow;
- Improved targeting of disease therapy
- More selective & efficient
- More efficient diagnosis of disease
- Disease markers
- Susceptibility genes
- Genes which make an individual more predisposed to a particular condition
- Delivery of personalised medicines
- Pharmacogenomics
- Pharmaceutical care of an individual based on their genetic make-up
- Pharmacogenomics
- Improved targeting of disease therapy
What is ‘omic’ technology and what does it aim to do?
- Aimed at the detection of biomolecules in a specific biological sample.
- Genes (genomics), mRNA (transcriptomics), proteins (proteomics)
- Enable the detection of therapeutically significant biomarkers.
- Underpin the current movement to transform health care from a model which is generalised and reactive, to one which is predictive, preventive and personalised.
What are important principles of nucleic acid detection methods?
- Base pairing rules are central to nucleic acid detection
- Need to understand the basis of DNA synthesis and the orientation of anti-parallel strands of DNA
- Need to consider levels of sensitivity, accuracy and reproducibility
Describe the difference in cell growth and the molecules involved.
What is cell growth controlled by?
- The different cells that exist in the body grow at different rates and in response to different stimuli.
- CDK (cyclin dependent kinases) and cyclin are key molecules that control and coordinate DNA-synthesis, chromosome separation and cell division
- CDK and cyclin together drive the cell from one cell cycle phase to the next.
- Cell proliferation is under the control of specific genes.
- Proto-oncogene: a cellular gene, usually encoding a regulatory protein, that can be converted into an oncogene by mutation.
- Oncogene: a cancer causing gene; any of several mutant genes that cause cells to exhibit rapid uncontrolled proliferation.
- The presence of oncogenes can therefore be both a target for treatment and a method of detection.
What phase of the cell cycle does DNA synthesis occur?
How is cell growth measured?
Measurement of synthesis of DNA gives an indication of cell proliferation
DNA synthesis occurs during the S phase of the cell cycle
In vivo: specific genes and proteins are expressed at key stages of the cell cycle
In vitro: add labelled nucleotide and determine how much is incorporated by the cells
Describe measurement of cell proliferation in vitro using labelled nucleosides?
In vitro labelled nucleotides are added to laboratory cell cultures and then actively taken up by cells involved in DNA synthesis and incoperated into DNA. These are then detected. Markers used:
- Bromodeoxyuridine (BrdU)
- AB specific to BrdU is used to detect BrdU and therefore incoperation of the nucleotide into DNA synthesising and therefore cell proliferation. There is a colout change
- Tritiated thymidine
- Overlay cells with film which is exposed by the radioactivity that hits film.
- Radioactivity emission is measured
- BrdU safer
How can DNA be damaged?
What are repair mechanisms for DNA?
Many conditions have a genetic component which predisposes them to being more likely to develop it (genome). However, there it is normally multifactorial and additional requires influence of other factors such as lifestyle, diet/nutrition and toxin exposure in order for this to develop and affect phenotype of cells.
Give and example of a condition like this and the factors which affect it
Describe the squence of DNA in context of genes
What is a single nucloetide polymorphism
What effects can this have on both the intron and exon regions of DNA?
What 2 methods are available to determine the sequence of DNA?
What does these help improve?
•Two methods developed in 1977 that greatly improved the efficiency and the accuracy of sequencing of DNA
– Maxam & Gilbert
– Sanger
What is the principle of SANGER’s method for DNA sequencing?
- Based on the controlled interruption of the enzymic replication of DNA i.e. synthesise DNA and know when it stops!
- The strand synthesis is initiated by a primer. The 3’ hydroxyl end of the primer will react with incoming dNTP and form a phosphodiester bond.
- SANGER used a di-deoxynucleotide (ddNTP) in addition to deoxynucleotides in the DNA synthesis reaction.
- The ddNTP has the same structure as dNTP apart from a -H at the 3’ end as opposed to the -OH. The incoming nucleotide requires the -OH in order to bind, therefore incoperation leads to halting of DNA
Describe the process of DNA sequencing using the SANGER method
SANGER method works by controlled interpuption of the enzymatic replication of DNA - i.e. synthesise DNA and know when it stops!
The strand synthesis is initiated by a primer and in normal DNA synthesis the 3’ hydroxyl end of the primer will react with incoming dNTP and form a phosphodiester bond.
However, the SANGER method incoperates di-deoxynucleotides (ddNTP) in addition to deoxynucleotides in the DNA synthesis reaction. These contain a -H at the 3’ end compared to a -OH. This -OH is required for the binding of the incoming dNTP, therefore this inhibits DNA synthesis.
To begin with the DNA is denatured i.e. strands separated by heating or alkaline conditions and a primer added which recognises specific sequences in the DNA. This allows for the initation of strand synthesis.
Enzymes and dNTPs added along with ddATP. The strand synthesis will therefore terminate where T is in the original strand when a ddATP is added.
As several ddATP are added and this will generate lots of different fragements of DNA of varying lengths depending on which T the ddATP interacts with.
Synthesis is carried out with each ddNTP (ddGTP, ddTTP, ddCTP) in a separate reaction to again produce fragments of varying lengths.
Each fragment is then seperated out using electophoresis. The fragments are run on agrose gel and an electrical current applied - as DNA is negatively charged the fragments will migrate to the +ve electrode. The fragements move as they are carried by the EOF. The smaller fragments travel the furthest.
Based on size/distance travelled and ddNTP added the sequence of DNA can be determined
Describe fluorscent labelling of individual nucleotides
Addition of fluorescent labels has sped up the rate at which this data can be analysed and gives far more accurate information.
Each ddNTP can be given a different colour so each one can be identified and used in the same reaction
Describe nucelic acid hybridisation
Principles of PCR
PCR Reaction mixtyre - essential ingreedients?
Key stages of PCR
- Denaturation - 2 strands break apart. Temperature depends on strands (more C/G higher as 3 H bond)
- Anealing - primers bind to target/ complementary sequence
- Extension and DNA replication occurs
Amplification of the product
Adv/Disadv of PCR
Can we use PCR for RNA?
Primer - sequence of T nucleotides so can attach to poly A tail on mRNA
Detection of mRNA
