Research techniques Flashcards
How does PCR occur?
In PCR non identical primers are annealed.
To opposite strands of the DNA template.
Always read from 5 prime end to the 3 prime end
What are the reagents required for PCR?
DNA polymerase
dNTPs
Buffers and salts
Primers
template
What are the advantages and disadvantages of PCR?
Advantages: Sensitive, quick, cheap, not labour intensive, do not require infectious material, specific.
Disadvantages: Contamination, Need target sequence information, primer efficiency, good primer design.
How do you determine PCR results?
Results are determined on a gel electrophoresis. Agarose gel. Smaller fragments going further. Larger fragments staying shorter.
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What are the two types of PCR?
Endpoint PCR: product is detected and visualised by agarose gel eletrophoresis at end of reaction.
Real-time PCR: allows quantification of products in real time. uses fluorescent based pcr. very sensitive.
Why does PCR amplify target sequence?
Specific primers are unique. Only one target site in the template DNA where the primers binds, which means the primer sequence should be unique in the template DNA.
What do you need when designing a primer and when doing pcr?
2 primers flanking either end of the sequence.
primer sequence must be complimentary to target DNA
Approx 15-28nt
Similar meltng temperatures in the pairs ideally between 5c and the Tm between 55-72c.
Stable base pairing not to many AT and too many GCs equal number prefarably
How do you calculate Tm?
- Wallace rule
Tm= 4x (G+C)+ 2x(A+T)
What do you try an avoid when making a primer?
1) Hairpin
2) Self-dimer
3) Dimer
What is immunohistochemistry and what does it do?
Is based on the binding of antibodies to a specific antigen.
It allows localisation of protien expression within tissue sections.
No specialist equipment just simple.
What are two main diffferent techniques in IHC?
Direct- Primary antibody is labelled directly with an enzyme or fluorescent dye.
Indirect- Unlabelled primary antibody binds to the target antigen in the tissue and a labelled secondary antibody is then added that reacts with the primary antibody.
Secondary antibody must be different to that of the primary antibdoy or else it will bind to the same thing and not the primary.
What are the advantages and disadvantages of IHC primary and secondary.
Primary- Adv. More specific
Disadv. Less sensitive
Seocndary- Adv. More sensitive as binding more
Disadv. Less specific as chance of binding to other proteins, also could be longer and more expensive process.
Name some markers for IHC, in particular T and B cells?
CD3- T lymphocytes
CD79a- B lymphocytes
vWF- Endothelial cells/ CD38
What are monoclonal antibodies?
They are made in mice
Have a lower sensitivity
High specificity
No batch variation
Can be expensive
What are polyclonal antibodies?
made in mutliple species
Higher sensitivity may bind to more than variance
Lower specificity may contain irrelevant antibodies if not affinity
batch variation as depends on the individual response to inoculation.
Can you name all the parts of the microscope?
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What is in situ hybridisation?
Method of localizing, either with mRNA within the cytoplasm or DNA within the chromosomes, by hybridizing the sequence of interest to a complimentary strand of a nucleotide peptide.
Nucleic acid hybridization is a fundamental tool in molecular genetics. It takes advantage of the complementary nature of double stranded DNA or RNA or even RNA to RNA.
Name some techniques of quantitative RNA analysis?
In situ hybridization
Northern blot
Real time PCR
Semi quantitative PCR
Lazer micro dissection PCR
microarray expression PCR
What are the advantages and disadvantages of the different techniques of quantitative RNA analysis?
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What probes are used for in situ hybridization?
Oligonucleotides-high specific
SS dna- reverse transcriptase
DS dna- denaturation necessary as only one strand is needed.
What has the strongest bond strength in the techniques?
- RNA- RNA
- RNA- DNA
- DNA -DNA
In probe labelling what is usual standard?
Direct- A nucleotide containing a fluorophore
Indirect- Chemical coupling of a modified reporter molecule. The reporter molecule can bind with high affinity to another ligand (biotin)
What does non radioactive ISH usually look like?
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What are the three main methods to label a probe?
Random prime labelling
PCR
In vitro transcription
How does in vitro transcription happen?
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Can you give an overview of how fluorescence in situ hybridization?
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