Introduction To Molecular Techniques Flashcards
Name techniques to analyse DNA at the gene level
Restriction enzymes DNA gel electrophoresis PCR Southern hybridisation Microarray PCR variations
Name some protein analysis techniques
Protein electrophoresis
Immunoassay
Enzyme assays
Name some techniques to analyse DNA at the nucleotide level
DNA sequencing
Name some techniques to analyse DNA at the chromosome level
Karyotyping
FISH
What does a restriction endonuclease do?
- recognition and degradation of foreign DNA
- specific endonucleases (restriction enzymes) recognise and cut specific DNA sequences (restriction sites)
- (mostly) palindromes of 4,5,6,8 bp
What is the purpose of gel electrophoresis?
To separate DNA fragments based on size
What are 4 requirements of gel electrophoresis?
- Gel - A matrix that allows separation of DNA fragments
- Buffer - Allows charge on the DNA samples across the gel and keeps pH constant
- Power supply Generates charge difference across the gel
- Stain/detection To identify the presence of the separated DNA
Will dNA move towards the anode or cathode if placed in an electric field?
Anode since DNA is negatively charged
Where will the smallest fragments be found?
The smaller the fragment the further it moves up the plate so the closer to the positive electrode
Outline the steps in electrophoresis
Put agarose in tray As it sets it forms a gel Make wells in one side Put DNA samples in the wells Electric field across Run DNA samples across from negative to positive Travel depending on size
Why use restriction analysis?
To investigate the size of DNA fragments
e.g. small deletions
To investigate mutations
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
- “mini-chromosomes”
- carry genes to
replicate independently - can transfer to other bacteria
- often carry antibiotic resistance genes
What are the 4 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
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 (replace abnormal genes with wild type)
Outline the steps in synthesis of proinsulin by bacteria
Obtain mammalian proinsulin mRNA
Use reverse transcriptase to obtain proinsulin complementary DNA (cDNA)
Join this gene to plasmid to create recombinant plasmid
Insert in e.coli to make transformed bacterium
What is the purpose of PCR?
Amplification of target DNA
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
Outline the steps in PCR
20 AA primer - complementary to end of sequence
Taq polymerase form thermophile bacteria
20-35 cycles
Heat mixture to 95C - denatures DNA breaking h bonds
Temp reduced to 60C - primers anneal (h bond)
Temp raised to 72C - optimum for taq - add nucleotide to 3’ end to each primer
After 1 cycle - 2 copies
What is Taq polymerase and why is it used in PCR?
DNA polymerase from thermophile bacteria, it is thermostable
Describe the nature of primers in PCR
Pair of primers (forward and reverse), uniquely
defining the region to be copied
What is protein gel electrophoresis?
Proteins are charged molecules and will move towards the anode or the cathode if placed in an electric field
How can proteins be separated in protein gel electrophoresis?
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 SDS-PAGE?
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Separates proteins based on size
Denature this protein
Add sds - detergent - read down
Add -ve charge proportional to length of protein
Gets rid of charge
What is Isoelectric Focusing (IEF)
Proteins separate on the basis of charge
Proteins migrate until they reach a pH equal to their pl
No net charge at pl so stop migrating
Briefly describe IEF
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 pl values
(Proteins have different Isoelectric points)
What is 2D-PAGE?
Two-dimensional gel electrophoresis
Allows the separation of complex mixtures of proteins
Important for diagnosing disease states in different tissues
Describe protein identification by proteomics
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
Why are antibodies useful for recognising proteins?
Antibodies bind to specific protein targets (antigens)
Recognise a few amino acids on a protein (epitope)
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 western blotting
A technique to detect proteins
Briefly describe western blotting
Nitrocellulose replica of gel electrophoretogram Primary antibodies raised against protein of interest Transfer proteins on nylon sheets Antibodies bind really tightly Wash Detect binding with second antibody Second antibody against primary antibody Immunoglobulin appears
What is ELISA?
Enzyme linked immunoabsorbent assay
Can be used to measure the concentration of proteins in solution e.g. Hormones, insulin, cortison, TSH
Describe indirect ELISA
Antigen coated well
Wash
Specific antibody binds to antigen
Wash
Enzyme linked antibody binds to specific antibody
Wash
Substrate is added and converted by enzyme into coloured product; the rate of colour formation is proportional to the amount of specific antibody
What are enzyme assays?
Measuring amount of enzyme present Mixture of enzyme and substrate Measure appearance of product over time Measure rate of enzyme activity International unit
Give 2 examples of continuous assays
Spectrophotometry
Chemoluminescence
Give 2 examples of discontinuous assays
Radioactivity
Chromatography
What can enzyme assays be used for
Can measure enzyme activity if we have a particular assay for it
Look for metabolic disorders
Why measure enzymes?
Metabolic disordered in tissues
Diagnosis of disease (serum enzymes)
What are some clinically important serum enzymes?
Alanine transaminase (ALT) (markers for liver damage/disease)
Amylase/ lipase (marker for pancreatitis)
gamma-glutamyl transferase (market for liver damage, increased by alcohol)
Alkaline phosphatase (marker for bone disorders)
