Molecular Techniques Flashcards
Requirements for gel electrophoresis: (4)
Gel - matrix allowing separation of DNA
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
What are restriction enzymes?
Molecular scissors.
They recognise and cut specific DNA sequences. Mainly palindromes. Come from bacteria
Why do we use restriction analysis?
To investigate the size of DNA fragments
To investigate mutations
To investigate DNA variation
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
Four basic steps of gene cloning:
Isolate relevant gene of interest following digestion with restriction enzymes
Insert gene of interest into plasmid vector
Introduce recombinant DNA molecule into suitable host cells
Identify and isolate clone containing DNA of interest
Why do we clone human genes?
To make useful proteins (insulin)
To find out what genes do
Genetic screening
Gene therapy
Steps of PCR:
Denaturation - DNA heated to 95 degrees
Annealing - the two primers bind the appropriate complimentary strand
Primer extension - DNA polymerase extends the primer. Taq polymerase works best at 72 degrees.
(Repeated 28-35 times)
Why use PCR?
To amplify a specific DNA fragment
To investigate single case mutations
To investigate small deletions or insertions
To investigate variation, genetic relationships
How are the proteins separated in electrophoresis?
Proteins are charged molecules and move towards the anode or cathode if placed in an electric field
Proteins are separated on basis of size, shape or charge
How is DNA separated in electrophoresis?
DNA is negatively charged and will move towards the anode of placed in an electric field
DNA fragments can be separated on the basis of size (or shape)
What does SDS-PAGE do?
Separation of proteins on the basis of size
What is Isoelectric focusing (IEF)?
Proteins separate on basis of charge
Proteins migrate until they reach a pH equal to their pl
No net charge at pl so stop migrating
Steps of Isoelectric focusing (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
Overview of 2D-PAGE
Allows separation of complex mixtures of proteins
Important for diagnosing disease states in different tissues
Proteomics steps
Digest protein with trypsin
Perform mass spectrometry
Generate list of peptide sizes
Use database of predicted peptide sizes for known proteins to identify the protein
Proteomics is
The analysis of all proteins expressed from genome
Molecular diagnosis is
Analysis of a single purified protein
Antibodies characteristics:
Bind to specific protein targets (antigens)
Recognise a few amino acids on a protein (epitope)
Monoclonal antibodies
Produced from 1 B Lymphocyte
I identical antibody
Specific to 1 antigen
1 epitope
Polyclonal antibodies
Produces by many B lymphocytes
Multiple different antibodies
Specific to 1 antigen
Multiple epitopes
What can an enzyme-linked immunoabsorbent assay (ELISA) be used for?
Can be used to measure the concentration of proteins in solution
What is the gold standard for diagnosis of an MI?
Cardiac troponin I (cTnI) by ELISA
What is southern blotting?
Combined gel electrophoresis and DNA hybridisation
What is northern blotting?
Uses DNA to detect RNA species. Combined gel electrophoresis and DNA hybridisation
What is western blotting?
Involves the detection of proteins by antibodies after protein gel electrophoresis
Steps of southern blotting:
Digest DNA with restriction enzymes
Separate DNA fragments by gel electrophoresis
Transfer DNA to nylon
Hybridise filter with labelled gene probe
Detect hybridisation by exposure to x ray film
Why do we use southern blotting?
To investigate gene structure
To investigate gene expansions, triplet repeats
To investigate mutations in genetic tests
To investigate variation, genetic relationships
Characteristics of DNA proves in binding:
Do not need 100% similarity to target sequence
Do not need to completely align with target
Do not affect the position of the target sequence on the gel
How does DNA sequencing work?
A tube contains all of the dNTPs, DNA polymerase and one of the ddNTPs.
It also has denatured DNA and a primer.
When the ddNTP is added the chain stops elongating.
This is done for all ddNTPs and then they are run on a gel and identified by labelling to read the sequence
Why would we use RT-PCR?
It reveals information about gene expression
Outline RT-PCR:
Nuclease breaks down the RNA to remove
DNA primers are used and anneal
Reverse transcriptase catalyses the reaction to extend the sequence
PCR is used to amplify the cDNA (complimentary)
What is microarray technology?
Analysis of 1000’s genes on a slide
Can compare two conditions
Can die the two slides with different probes. You can compare gene expression depending on the size of the signal
You can see duplications and deletions
What is chromosome painting?
Making proves specific for each chromosome.
Can be helpful to identify translocation and can be used for cancer cell analysis
What is FISH?
Probe DNA is labelled with fluorescent dye
Target chromosome denatured and hybridised
Re annealed and can be viewed
