Lecture 6 (Lecture 5 Was About Assessment) Flashcards
What determines the tissue structure and function in the body?
Gene expression —> protein composition —> cell(tissue) structure and function
What are some of the things that can be seen using molecular pathology techniques?
- Gene amplifications
- Gene mutations
- Translocations
- Chromosomal abnormalities
- Gene deletion
Wha are some examples of molecular pathology techniques that are used?
- PCR
- DNA sequencing
- Cytogenetics
- Southern blot, norther blot and western blot
- Microarray
- Melting curve analysis
- ISH (in situ hybridisation)
- Autoradiography
How do we quantify DNA?
- Gel electrophoresis
- Spectrophotometric determination (nanodrop)
How do we amplify DNA?
PCR
Look up reverse transcription PCR if haven’t done already in genetics
Yeah boi
What is DNA microarray?
- Has hundred of probes (ssDNA) on the microscope slide which targets complimentary strands that are in the sample (cDNA)
- The cDNA and the ssDNA binds to form hybrid double helix (DNA hybridisation)
What does microarray use?
- It uses ‘grids’ on a substrate
- Each grid features or addresses a particular gene or sequence of DNA (containing 100-1000s of identical probes)
- Each array run contains 1000s of grids each looking for a specific gene sequence
- The grids can be compared against each other to see how the genes are being expressed
How are DNA microarrays analysed?
-Use a gene ontology software which interprets and groups data because its too much for humans to try and sort and interpret
What is in situ hybridisation and how is it used?
Similar to immunohistochemistry in the way that it works
- DNA or RNA probes are used to detect a specific sequence of DNA or RNA can be visualised in the tissue via a radioactive or fluorescent label
- Can be used to show chromosome enumeration
- Visualise specific gene responses
- Can be used during cancer diagnosis to find the growth factor (HER2)
What are the northern, southern and western blots?
- Southern blot - means of detecting the presence and detection of DNA fragments within tissue samples
- They are all methods of transferring DNA, RNA or proteins to a carrier (eg nitrocellulose)
- Usually performed after Gel electrophoresis
- Visualised by a colourant staining
What is the southern blot method?
- Used for the detection of specific DNA sequences in a sample
- Used electrophoresis separated DNA fragments combined with a filter membrane
- This membrane can then be exposed to a specific hybridisation probe
- This is visualised then through and X-ray film or through fluorescence
What is northern blot and how is it used?
- Used to study gene expression by the detection of RNA
- RNA is extracted and electrophoresis is separated and placed on either nitrocellulose or nylon membranes
- The membranes are then visualised via X-ray, fluorescence or using a special dye
What is the western blot and how does it work?
- Used to detect specific proteins in a sample of tissue or extract
- It detects protein in the sample via molecular weight
- It uses antibodies to bind to specific antigen epitopes in tissue
- This can be visualised on a film using various detection methods eg (Chemiluminescent, radioactive probe or fluorescence)
What are some of the uses of western blot?
- Use for the detection of anti-HIV antibody in a human serum sample
- It is used as a definitive test for CJD
- Use in detecting Lyme disease
- Also used as a confirmatory test for hepatitis B and HSV-2 (herpes type 2) infection
