Nucleic acids 6- analysis of nucleic acids Flashcards
Describe the role of personalised medicine in HER-2 receptor positive breast cancer
Overexpression of HER2 leads to more aggressive breast cancer
(involved in cell growth and proliferation)
20% of breast cancer is Her2 positive- more aggressive
Drugs (biologic therapy) have been developed specifically for
cancer where individuals have a Her2 mutation
e.g. Trastuzumab (Herceptin)
What are the clinical advantages of next generation sequencing
Allows us to introduce more targeted therapies- does the patient have the HER-2 mutation or not.
More practical- hand-held device
What is DNA cloning
A method of selectively amplifying DNA sequences of interest to generate homogenous DNA populations
Cell-based DNA cloning (in vivo)
Cell-free DNA cloning (in vitro) - PCR
What is a replicon
A sequence capable of independent replication (a plasmid, bacteriophage, Yeast Artificial Chromosome)
Why do we need to cut the genome into fragments before cloning
The genome is too long and unwieldy to be handled easily in the laboratory- hence it needs to be cut into more manageable pieces.
Describe cell-based DNA cloning
cutting a target DNA and a replicon, with specific enzymes, restriction endonucleases, so that the ends of the two DNA sequences are compatible.mixing and joining the DNA fragments by using the enzyme DNA ligase. Transformation of the recombinant DNA molecules into host cells (bacteria, yeast.Selective propagation of individual cell colonies on agar plate.selectable antibiotic resistance marker in the replicon; only cells with replicon survive)
Expansion of the cell culture and isolation of recombinant DNA- culture in a growth medium.
What is the role of the replicon in DNA cloning
It is the vector-
What are restriction endonucleases
Type II restriction endonucleases are enzymes that cleave DNA at specific recognition sequences
Recognition sites are usually 4-8bp palindromic sequences
Describe the two types of cut that restriction endonucleases make
Blunt- cut at the point of symmetry
Staggered cuts- create sticky ends- short, single-stranded overhangs that help the cut DNA molecules join back together by Complementary base paring with, with the aid of ATP and DNA ligase to reseal the sugar phosphate-backbone.
Where are restriction endonucleases obtained from
Restriction endonucleases are one half of bacterial restriction-modification systems, a kind of primitive immune system. Host DNA is protected by methylation of a base in the RE site by a specific methylase - RE will only cleave unmethylated DNA from invading organisms, not host DNA
If the recognition site is longer is it likely to occur more frequently or less frequently within the genome
Less frequently
How are DNA fragments separated and how does this work
Electrophoresis
DNA is negatively charged due to its phosphate backbone and it moves towards the anode (+ve electrode) when an electrical force is applied to a DNA solution
Describe how the DNA fragments are separated based on size
DNA size resolution
When DNA is forced to travel through a porous gel matrix (agarose / polyacrylamide gel) small fragments are retarded less than large fragments and hence travel faster
How do we isolate the desired fragment
Excise it with a scalpel or blade
How can we visualise DNA fragments
Expose the gel to a dye that fluoresces under UV light when it is bound to DNA- when placed in a UV box, the individual bands glow bright orange, or bright white when the gel is photographed in black and white.
Incorporate a radioisotope- beta particles emitted from the P-32 can activate the radiation-sensitive particles in photographic film, a sheet of film placed on top of the agarose gel will, when developed, show the position of all the DNA bands..
What is the purpose of nucleic acid hybridisation
A method for detecting specific nucleic acid sequences in which homologous single-stranded DNA or RNA molecules combine to form double-stranded molecules
What is a standard assay in hybridisation
Standard assay involves a labeled ( * ) nucleic acid probe to identify homologous related target molecules in a mixture of unlabeled nucleic acids
Describe southern blot hybridisation
Target DNA (e.g. from an agar plate or gel) is immobilised on a solid support - nylon or nitrocellulose membrane - which readily binds single-stranded nucleic acid (e.g. denatured DNA or mRNA) and then hybridised with a solution of (radioactively or fluorescently) labeled (*) probe
How is the DNA denatured in southern blotting
By the presence of an alkali solution, buffer, drawn towards paper towels, carrying the singl-stranded target DNA to the nitrocellulose paper where it is then immobilised.
Why do we wash the nitrocellulose sheet after adding the probe
So that only probe molecules that have hybridised to the DNA on the paper remain attached.
Describe the variation in hybridisation assays
Southern blot hybridisation (DNA target and DNA probe)
(after Ed Southern)
Northern blot hybridisation (RNA target and DNA probe)
Colony blot hybridisation (bacterial DNA target, DNA probe)
Tissue in situ hybridisation (RNA target and RNA probe)
Chromosome in situ hybridisation (Chromosome target and DNA probe)
Reverse hybridisation – Microarrays (immobilised DNA or oligonucleotide probe, target DNA solution)
Describe the purpose of in situ hybridisation
Single-stranded DNA or RNA labelled probes detect complementary DNA or RNA within a tissue or cell, or even on a chromosome, showing which genes are expressed.
How do we denature probe DNA, and what does the energy to do this depend on
Denaturation of a probe DNA is achieved by heating until the hydrogen bonds between the bases holding the two strands together are disrupted.
The energy needed to do this depends on:
Strand length: longer strand = more hydrogen bonds to break
Base composition: G-C pair has one more hydrogen bond than A-T, so harder to break
Chemical environment:
Monovalent cations (Na+) stabilise the DNA duplex by neutralising charge on phosphate backbone
denaturants (formamide / urea) destabilise the DNA duplex
What is the melting temperature (Tm)
Melting temperature (Tm) - measure of nucleic acid duplex stability. Midpoint temp. of transition from double stranded (DS) to single stranded (SS) forms of nucleic acid
For mammalian genomic DNA (40% GC) this is ~87oC
Hybridisation is carried out at temperatures <25oC below Tm- to ensure a 50:50 mix of DS and SS DNA, so that hybridisation can occur.
What is hybridisation stringency
Hybridisation stringency (i.e. the power to distinguish between related sequences) increases with:
Increase in temperature
Decrease in Na+ concentration.
reaction conditions, notably temperature and salt, that dictate the annealing of single-stranded DNA/DNA, DNA/RNA, and RNA/RNA hybrids
at high stringency, duplexes form only between strands with perfect one-to-one complementarity
lower stringency allows annealing between strands with some degree of mismatch between bases
Describe cell-free DNA cloning
Polymerase Chain Reaction (PCR)
In vitro method to allow selective amplification of a specific target DNA within a heterogeneous collection of DNA sequences (e.g. total genomic DNA or complex cDNA population)
What are the three requirements for PCR.
Some sequence information of the target (e.g. gene of interest) is needed to design 2 primers (15 - 25 nucleotides in length), one complimentary to each strand of the DNA to be copied (“forward” and “reverse”)
DNA polymerase can only extend and existing chain. We need two primers, as chain extension can only proceed in 5’-3’.
We need the two strands of the DNA double helix to be anti-parallel
What are the advantages of PCR
Quicker, can amplify even tiny samples- useful in crime scene
Selective- although original irrelevant DNA is still present, the cloned DNA is in such an excess that we assume the final product to be a slightly impure preparation of the target sequence
Describe the process of PCR
Primers are specifically annealed to heat denatured (95) DNA by lowering temperature (50-60)
Thermostable Thermophilus aquaticus DNA polymerase + dNTPs extend 5’->3’ from the primers and generate new strands- 72- optimum temp for Taq Polymerase.
This cycle is then repeated around 30 times
What does the selectivity of PCR depend on
Having primers that will only anneal to the desired target. This means that having short primers is ideal, and fine-tuning the temperature is essential, too low and the primers may be able to hybridise with less stringency- computer programmes assist with primer design to ensure that they are only complementary to the target region of DNA.
Length
Usually about 20 nucleotides for a complex genomic DNA target – gives required specificity for target sequence
Base composition
Avoid tandem repeats of nucleotides that can form hairpins
%GC and length should give an ~equal Tm for each primer
3’ end
Avoid complementarity of the bases at the 3’ ends
Primer dimers may result
What are the applications of PCR
Typing genetic markers (Restriction Fragment Length Polymorphisms)
Detecting point mutations
restriction site changes, Allele-specific amplification
cDNA cloning
Genome walking
Gene expression – Reverse Transcription-PCR (reverse transcribe mRNA to DNA using oligo-dT [binds to polyA tail], then amplify)
Introducing mutations experimentally (base mismatches)
DNA sequencing
DNA microarrays
What are DNA (oligonucleotide) arrays
A DNA (or oligonucleotide) microarray is a collection of microscopic DNA (or oligonucleotide) spots, commonly representing single genes, robotically arrayed on a solid surface, e.g. a glass slide
Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridisation under high-stringency conditions, with fluorophore-based detection
DNA arrays are commonly used for expression profiling, i.e. monitoring expression levels of thousands of genes simultaneously, or for comparative genomic hybridization
Why doesn’t the target sequence rehybridize to the original sequence
it may, but the primers are present in much greater numbers.
What are the applications of microarrays
mRNA or gene expression profiling
- monitoring expression levels for thousands of genes simultaneously is relevant to many areas of biology and medicine, such as studying treatments, disease, and developmental stages. For example, microarrays can be used to identify disease genes by comparing gene expression in diseased and normal cells
SNP detection arrays - looking for Single Nucleotide Polymorphisms in the genome of populations
Describe simply the process of microarray
cDNA probe binds to mRNA from cell- disadvantage of only showing expression of genes targeted by the probe- different colours represent different levels of gene expression- reverse hybridisation.
