Session 12-Intro To Molecular Techniques Flashcards
What are the five ways in which DNA can be analysed at a gene level?
Restriction enzymes DNA gel electrophoresis PCR variations Southern blotting Microarrays
What are the three ways in which proteins can be analysed?
Protein electrophoresis
Immunoassays
Enzyme assays
How is DNA analysed at a nucleotide level?
DNA sequencing
What are the two ways in which DNA can be analysed at a chromosome level?
Karyotyping
FISH
Why do bacteria produce endonucleases?
To recognise and degrade foreign DNA
Which restriction site does the restriction enzyme EcoRI recognise?
GAATTC
How does DNA gel electrophoresis separate fragments?
Depending on their size
Why does DNA move towards the positive electrode in gel electrophoresis?
DNA is negatively charged (PO4 3-)
What are the requirements for gel electrophoresis? (4)
- Gel
- Buffer
- Power supply
- Stain/detection (fluorescent markers)
Why use restriction analysis? (4)
- To investigate the size of DNA fragments
- To investigate mutations eg. Sickle cell disease
- To investigate DNA variation
- To clone DNA
What are plasmids?
Small circular dsDNA found in bacteria which carry genes to replicate independently
What are the four basic steps of gene cloning?
- Isolate relevant gene of interest following digestion with restriction enzymes
- Insert gene of interest into plasmid vector (recombinant DNA molecule)
- Introduce recombinant DNA molecule into suitable host cells eg. E. coli
- Identify and isolate clone containing DNA of interest
Why are human genes cloned? (4)
- To make useful proteins eg. Insulin
- To find out what genes do
- Genetic screening
- Gene therapy
What does reverse transcriptase use as a template for DNA?
RNA
What is PCR used for?
- DNA amplification
- To investigate single base mutations eg. Tay Sachs, Sickle cell
- To investigate small deletions or insertions eg. Cystic fibrosis
- To investigate variation, genetic relationships
What is Taq polymerase?
DNA polymerase derived from hot springs bacteria- can withstand the high temperature of PCR
Describe the process of PCR (5)
- Amplification of target DNA
- Thermostable DNA polymerase (Taq)
- Pair of primers (forward and reverse), uniquely defining the region to be copied
- Temperature cycles of denature, anneal and polymerise
- Repeated copying results in exponential increase in DNA
On what basis are proteins separated in protein gel electrophoresis?
Size, shape or charge
What is the difference between DNA and protein gel electrophoresis?
Protein g.e. Uses a vertical, rather than horizontal, method
What is native electrophoresis?
Using the intrinsic properties of the protein to separate it
What are the methods of separating proteins on the basis of size called?
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
Two-dimensional electrophoresis (2D-PAGE)
What happens in SDS-PAGE?
The 3D structure of the protein is broken, the protein is made negative and the disulphide bonds are broken i.e. The secondary and tertiary structure of the protein is gotten rid of so the protein size is known
How are proteins separated on the basis of charge?
Isoelectric focusing (IEF)
What happens in IEF? (3)
- A stable pH gradient is established in the gel after application of an electric field
- Protein solution is added and electric field is reapplied
- After staining, proteins are shown to be distributed along pH gradient according to their pI values
Describe the process of protein identification (proteomics) (4)
- Digest protein with trypsin
- Perform mass spectrometry
- Generate a list of peptide sizes
- Use database of predicted peptide sizes for known proteins to identify the protein
What is the difference between proteomics and molecular diagnosis?
Proteomics = analysis of all proteins expressed from genome
Molecular diagnosis = analysis of a single purified protein
Which mnemonic is used for the different types of blots and what they are used for?
SNOW DROP:
S - southern blotting corresponds to D (DNA)
N - northern blotting corresponds to R (RNA)
Os cancel out
W - western blotting corresponds to P (protein)
Which method is used to measure the concentration of proteins in a solution?
Enzyme-linked immunoabsorbent assay (ELISA)
Describe the steps of ELISA
- Antigen-coated well
- Specific antibody binds to antigen
- Enzyme-linked antibody binds to specific antibody
- Substrate added and converted by enzyme into coloured product; rate of colour formation is proportional to amount of specific antibody
What is the difference between ELISA and radioimmunoassays?
Radioimmunoassay works in the same way as ELISA but uses a radio-labelled primary antibody
Name two continuous enzyme assays
Spectrophotometry
Chemoluminescence
Name two discontinuous enzyme assays
Radioactivity
Chromatography
What are the serum enzyme markers for liver damage/disease?
Aspartate transaminase (AST) Alanine transaminase (ALT)
What is the serum enzyme marker for pancreatitis?
Amylase/lipase
What is the serum enzyme marker for bone disorders?
Alkaline phosphatase
What is the standard for diagnosis of an MI?
Measurement of cardiac troponin I (cTnI) by ELISA
Give an example of when enzymes are used to measure clinically important metabolites
Measurement of glucose concentration with glucose oxidase:
Test strips-H2O2 converted to coloured dye
Glucose monitor-glucose oxidase used as biosensor
Why do proteins move in gel electrophoresis?
Proteins are charged molecules and will move towards either the anode or cathode if placed in an electric field
