17. Intro to Molecular techniques Flashcards
What can be used for analysis of DNA at the gene level?
- restriction enzymes
- DNA gel electrophoresis
- PCR
What can be used for analysis of proteins?
- protein electrophoresis
- immunoassays
- enzyme assays
What are restriction enzymes?
specific endonucleases that cut phosphodiester bonds in both strands within SPECIFIC DNA sequences (restriction sites)
Where are restriction enzymes made?
Bacteria produce endonucleases
What do restriction enzymes commonly work on?
Palindromes of 4,5,6,8 bp
What type of ends do restriction enzymes leave?
Sticky ends
Define overhang.
Single stranded DNA produced at the site of cut by restriction enzymes.
How does gel electrophoresis work?
- A solution of DNA molecules is placed in gel with electric field across it
- DNA is negatively charged due to phosphate groups so moves towards the positive electrode
- Small DNA molecules move more quickly through the gel than larger DNA molecules.
- DNA fragments can be separated on the basis of size (or shape)
What are molecular weight size markers?
set of standards that are used to identify the approximate size of a molecule run on a gel.
What is used to stain DNA in gel electrophoresis?
Ethidium bromide (flat, hydrophobic: sits between bases) - made visible by shining UV light.
What are the requirements for gel electrophoresis?
- Gel: A matrix that allows separation of DNA fragments
- Buffer: Allows charge on the DNA samples across the gel
- Power supply: Generates charge difference across the gel
- Stain/detection: To identify the presence of the separated DNA
Why use restriction analysis?
- To investigate the size of DNA fragments
e. g. small deletions - To investigate mutations - affects restriction sites and so alters size of fragments
e. g. Sickle Cell disease - To investigate DNA variation
e. g. DNA fingerprinting - To clone DNA
What are plasmids?
- small circular dsDNA
- found in bacteria
- carry genes to replicate independently
- can transfer to other bacteria
- often carry antibiotic resistance genes
What are four basic steps in 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 e.g. E. coli
- Identify and isolate the clone containing the DNA of interest
What must be done before inserting DNA into plasmid?
Bacteria have no mechanism to remove introns:
- mature mRNA converted back into DNA
- using reverse transcriptase from viruses
Why clone human genes?
- To make useful proteins
e. g. insulin - To find out what genes do
e. g. HTT - Genetic screening
e. g. Huntington’s, BRCA1/2, Cystic Fibrosis - Gene therapy ?
e. g. Cystic Fibrosis
What does PCR stand for?
polymerase chain reaction
How does PCR work?
Denaturation:
- DNA is heated to 95C to denature and separate the strands by breaking hydrogen bonds.
Annealing:
- Temperature cooled to 50-65C to allow primers (that are complementary to the strands) to anneal
Elongation:
- Temperature raised to 75-80C to allow polymerisation and add to the primer (free nucleotides needed)
What enzyme is commonly used in PCR?
Taq polymerase because it is thermostable.
What does PCR do?
Amplifies DNA fragments
What apparatus is used to produce the required temperatures in PCR?
Thermocycler.
What are the primers used in PCR called?
Forward primer and reverse primer
Why use PCR?
- To amplify a specific DNA fragment
- To investigate single base mutations
e. g. Tay Sachs, Sickle Cell disease - To investigate small deletions or insertions
e. g. Cystic Fibrosis - To investigate variation, genetic relationships
e. g. DNA profiling, DNA typing
What is protein gel electrophoresis?
- Proteins are charged molecules and will move towards the anode or the cathode if placed in an electric field
- Proteins can be separated on the basis of size, shape or charge
What are the requirements for protein gel electrophoresis?
- Gel: A matrix that allows separation of the protein sample
- Buffer: Maintains charge on the protein samples
- Power supply: Generates charge difference across the gel
- Stain/detection: To identify the presence of the separated proteins
What is an SDS-PAGE?
Sodium dodecyl sulphate polyacrylamide gel
electrophoresis.
What is used in an SDS-PAGE?
- Beta Mercaptoethanol
- Sodium dodecyl sulphate (SDS)
- Polyacrylamide gel (PAGE)
What does an SDS-PAGE allow separation of proteins by?
Just by SIZE:
- SDS is a detergent which breaks down secondary structure and adds negative charge (removes intrinsic charge)
- all proteins have negative charge - so just separarting by size
- b-ME breaks disulphide bonds
- stained
- compare to standard sizes of molecules to determine what a molecule is
what is Serum protein electrophoresis
Assessing serum proteins - separated by size and charge
How is proteomics done?
A. Digest protein with trypsin B. Perform mass spectrometry C. Generate list of peptide sizes D. Use database of predicted peptide sizes for known proteins to: E. Identify protein
What is isoelectric focusing?
A specialised form of electrophoresis where proteins are separated on the basis of isoelectric pH.
How does isoelectric focusing work?
- A gel with a pH gradient is loaded into a tube.
- A charge is run through the gel
- proteins move through the gel until they reach the pH that is equal to their isoelectric point.
What do antibodies bind to?
Antigens.
What is proteomics?
study of all the proteins - Protein identification
Define epitope
the part of an antigen molecule (few amino acids) to which an antibody attaches itself.
What are the two different types of antibody?
Polyclonal and monoclonal
What are polyclonal antibodies?
- Produced by many B lymphocytes
- Multiple different antibodies
- Specific to 1 antigen
- Multiple epitopes
What are monoclonal antibodies?
- Produced from 1 B lymphocyte
- 1 identical antibody
- Specific to 1 antigen
- 1 epitope
What is a western blot?
Separation of proteins via electrophoresis, followed by identification of specific protein by binding of primary antibody and an enzyme linked secondary antibody
What is the ELISA test?
Enzyme-Linked Immunosorbent Assay:
- antigen coated well
- specific antibody binds to antigen
- enzyme linked secondary antibody binds to specific antibody
- substrate is added and is converted by enzyme into coloured product
(wash in between each stage)
Rate of colour formation is proportional to the amount of specific primary antibody
What is a continuous enzyme assay? give examples.
one assay giving the rate of reaction
e.g.
• Spectrophotometry
• Chemoluminescence
What is a discontinuous enzyme assay? give examples.
samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples
e.g.
• Radioactivity
• Chromatography
What are enzyme assays and what are they used for?
- measure the rate of enzyme activity in tissues or serum
- can get standard rates and compare in metabolic disorders to diagnose diseases linked with low activity of enzymes
- can see if enzymes are in the wrong place —> diagnosis of disease
- can detect enzyme elevations after acute MI
Give an example of when serum protein electrophoresis is used?
In monoclonal gammopathies e.g. multiple myeloma —> see changes in amount of globulins/ albumin —> big increase in gamma globulin
What are ELISA tests used for?
Measure the concentration of proteins in solution e.g. hormones - insulin, cortisol, TSH
The more Ab that binds the more protein that is present
What important enzymes are markers for liver damage?
AST ALT
Gamma-glutamate transferase (increased by alcohol)
What enzymes are markers for pancreatitis?
Amylase lipase
What enzyme is a marker for bone disorders?
Alkaline phosphatase
What can follow PCR?
- restriction analysis
- gel electrophoresis
- DNA sequencing
- southern blotting (probe)
- another PCR - nested PCR
What is the open reading frame?
includes the start codon, the part that codes for amino acids and the stop codon - introns removed
What mutation in sickle cell anaemia destroys a restriction site?
Single base substitution
A —> T converting Glu to Val
Destroys MstII restriction site so restriction enzyme wont cut it
What chromosome and gene is mutated in sickle cell aneamia?
HBB gene chromosome 11
Can PCR be used on RNA? What is the alternative, if any?
No, taq polymerase does not recognise RNA template
Alternative: take out mRNA - convert to cDNA - then to DS DNA to use as a template for PCR
What are primers in PCR and what is their function?
- small section of DNA complimentary to a certain sequence of DNA - anneal to sequence to show Taq DNA polymerase where to bind for amplification
