Laboratory techniques Flashcards
What are the uses of PCR?
- Make more DNA
- Determine if DNA is present
- Determine amount of DNA (how quickly does it amplify)
What are the ingredients in PCR?
- Sample (DNA)
- DNA polymerase
- Primer
- NTs
Describe one PCR cycle?
Heat sample
- DNA denatures into SS
Cool sample
- Primer anneals (binds) complementary DNA
Warm sample
- DNA polymerase elongates from primer
Repeat
What is the purpose of adding fluorescent dye to PCR?
Means you can quantify how much DNA is in a sample based on how quickly the DNA becomes florescent
How can PCR be useful in HIV?
cDNA is amplified
- Amount of cDNA produced over time will indicate VIRAL LOAD
What else is required in HIV PCR?
Reverse transcriptase to make cDNA
What can PCR be used to diagnose?
Herpes simplex encephalitis
What different blots identify what molecular structres?
- Southern blot: DNA
- NoRthern blot: RNA
- Western blot: Proteins
West produces proteins
South America has mixed DNA, Edward Southern invented it
What identifies DNA in a sample in the Northern blot?
Probe
- Single stranded DNA molecule, carries radioactive or chemical markers
- Once bound the markers reveal DNA sample
What are the steps involved in the southern blot technique?
- DNA sample enzymatically cleaved by restriction nucleases
- Pieces put on gel electrophoresis, seperated by size
- Blotting then occurs, filter paper put over
- Probe added, unbound probe washed away, leaving only bound probe, filter paper exposed to film which will reveal the bound DNA
What is RFLP (Restriction fragment length polymorphisms) analysis?
Describe its purpose and what the technique involves?
Technique used to characterise genes
- Restriction endonucleases used
- Analysis of fragments can reveal what genes are present
- Different genes have different lengths
- Southern blotting then used to detect lengths after fragmentation
What do Restriction endonucleases do?
DNA cutting enzymes - cut DNA at specific base sequences
Restriction fragment lenght piolymorphism technique is useful in what?
Working out inheritance of different alleles
What disease can be diagnosed through RFLP (Restriction fragment length polymorphisms) analysis?
Sickle cell
- Different Beta globin genes will have different lengths
Northern blot is useful for assessing what?
mRNA levels (gene expression)
What is used instead of a probe in Western blot technique?
Specific antibody to protein
What diseases may be detected through antibodies with Western blot?
- IgG or IgM in Lyme disease
- IgG HIV-1
What is the SouthWestern blot used to study?
DNA-protein interactions
- Especially transcription factors
Describe the SW blot
Proteins seperated by electrophoresis
- DNA then added as a probe - to see if they interact
What is flow cytometetry?
Analysis of cells as they flow through a liquid through a narrow steam
- Used to analyse cells by size and surface proteins
What are the key components of a flow cytometer?
- Flow cell: moves cells through machine
- Laser: light scattered by cells
- Photodetector: detects light scatter
What will the forward scatter in flow cytometetry detect?
Size of cell
What will the side scatter in flow cytometetry detect?
Granularity
What is antibody staining with flow cytometery useful for?
Presence of preotein in cells
What are the specific antibodies tagged with in antibody staining?
Unique fluorochrome
- Flow cytometer will detect fluorochrome if bound to cell
How can flow cytometry be used to detect fetal maternal hemorrhage?
Monoclonal antibody to hemoglobin F
- Detects fetal hemoglobin in red cells of the maternal circulation
What do fluorescently-labeled monoclonal antibodies bind to on RBCs?
GPI (glycosylphosphatidylinosito) anchored proteins
What disease will show reduced or absent RBCs after flow cytometry?
Paroxysmal nocturnal hemoglobinuria
- This is because they do not contain GPI anchored proteins which is what antiBs bind to
What are some examples on GPI anchored protiens?
- Decay accelarating facgtor (DAF/CD55)
- MAC inhibitory protein (CD59)
What does ELISA (Enzyme-linked immunosorbent assay test detect?
Antigens and antibodies in the serum
What are the different forms of ELISA test?
- Direct
- Indirect
- Sanwich
- Competitive
What are the steps involved in the direct ELISA test?
Add serum to plate -> antigen secured to surface
- Wash away liquid
- Enzyme-labeled antibody specific to antigen added
- Wash away unbound antibodies
- Substrate added -> colour change
- Enzyme-linked antibodies directly bind to the antigen
What is the indirect ELISA test involve?
Serum added for analysis
- Antibody added and binds to antigen
- Antibody and not enzyme linked
- Add an enzyme-labeled secondary antibody
- Substrate added -> colour change -> identification of antigen
What type of ELISA test requires a second antibody?
Indirect ELISA
- Secondary antibody is enzyme-linked - which will show colour change
What is more commonly done the direct or indirect ELISA test?
Indirect
What type of ELISA test requires a capture antibody first which the antigen then binds to?
Sandwich ELISA
- Antigen sandwiched between the 2 antibodies
- Can be direct or antigen
What are the advantages of the sandwich ELISA test?
- High specificity
- Works with complex samples (antigen does not require purification)
- Can use secondary antibody like indirect
What is the competitive ELISA test?
Primary antibody incubated with the sample
- Antigen-antibody complexes form
- These are washed away
- Secondary antiB and substrate will be added
- More antigen = more binding = less free antibody
-
What does a bigger colour change indicate in competetive ELISA test?
LESS antigen in sample
What can ELISA be used to detect?
HIV antibody (indirect)
What antigen can be detected to identify HIV on ELISA?
HIV p-24 antigen
- Often sandwich ELISA used
What can a DNA Microarray (chip or microchip) contain?
Glass, plastic or silica
What does DNA MIcroarray involve the use of to identify a sample?
Thousands of probes
- Used to test a sample of DNA with fluorescent markers
- Sample hybridizes with complementary bases
- Computer detects which probes bind sample
How can DNA Microarray be used to identify gene expression?
Cellular mRNA can be collected and converted to cDNA
- cDNA can be tested using microarray
- Can then determine which genes have been expressed
Why may we want to identify gene expression? (using DNA microarray)
Give an example
Cancer
- Which genes are active vs inactive
- Cancer cells vs normal cells
What is copy number variation and when can it be useful?
Some cells contain incr/decr copies of genes DNA
- Increased/decr copies linked to disease and can be identified via an increased or decreased fluoresence
What are SNPs?
Single Nucleotide polymorphisms
- Genes with variations in a single nucleotide
- Variations represented in microarray
- Binding indicates which SNP present in sample gene
- Many SNPs associated with disease and also preserved within families
Describe FISH (Fluorescent in situ hybridisation) What can it identify
- Fluorescent DNA probe binds to specific gene site
- Localises genes to a chromosome
- Can determine which chromosome contains a gene
What is hybridization?
Binding of probe to complimentary DNA/RNA
What part of the cell cycle is FISH mostly done in?
Metaphase
- Cells arrested in mitosis so chromosomes are visible individually
What can FISH identify?
Chromosomal abnormalities (test cells compared to normal cells)
- Microdeletions as no fluoresence on chromosome (DiGeorge)
- Translocation
- Duplication (extra site of fluorescence)
