Molecular Techniques And Diagnosis Flashcards
What is southern blotting?
Use gel electrophoresis to separate fragments of DNA (digested by endonucleases)
Soak in alkali to denature DNA
Then blot them onto a membrane nylon/nitrocellulose (capillary action)/electrophoretic transfer)
Then add labelled DNA probes
Wash
Visualise probes by exposing to X Rays.
What can denature DNA?
Heat, or pH>7
How are probes made?
Cloned DNA
Or made in the lab by an oligonucleotide synthesiser
Why use southern blotting?
Identify DNA Sequences from complex mixtures
Detect very small amounts of DNA
What can southern blotting be used for?
Investigate gene structure - large insertions or deletions
Investigate gene expansions and triplet repeats like in Huntingdonshire and fragile X syndrome
Identify mutations using allele specific probes
Investigate variation and relationships - genetic fingerprinting.
Can a probe that is not 100% complementary bind to DNA?
Yes, but it will bind less tightly.
What is the Sanger chain termination method?
Dideoxy chain termination
Uses dideoxynucleotides - no 3’ OH
If it is incorporated into growing DNA it prevents further elongation.
Have four test tubes, each containing a denatured DNA template, all 4 deoxynucleotides and one dideoxynucleotide, a primer (labeled) and DNA polymerase.
Incubate at 37 degrees Celsius
Gel electrophoresis in different lanes.
Read off the sequence
Now use fluorescently labelled dideoxynucleotides, all in the same test tube - produce chromatogram.
What produces endonucleases and why?
Bacteria, to recognise and degrade foreign DNA.
What do endonucleases recognise?
Recognise specific DNA sequences (usually palindromic, and 4,5,6 or 8 base pairs long)
What is the result when endonucleases digest DNA?
Fragments with over hanging single stranded DNA (sticky ends)
What produces EcoRI? Where does it cleave?
Escherichia coli strain RY13
GAATTC, cleaves between G and A
How do bacteria protect their own DNA from endonucleases?
Methylation
How many different restriction sites have been identified?
More than 200!
What is the gel in DNA gel electrophoresis?
Agarose
Where are the electrodes found in DNA gel electrophoresis?
Negative electrode at the wells for sample loading where larger fragments remain
Positive electrode at the other end, attracts negatively charged DNA.
What is required for gel electrophoresis?
Buffer
Gel
Power supply
Stain
Why use restriction analysis?
Investigate the size of fragments, identify small deletions
Investigate mutations
DNA variation and fingerprinting
Clone DNA.
How can genes be rejoined?
Cut with restriction enzymes
If overhanging sequences are complementary, can join with DNA ligase.
What are plasmids?
Small circular double stranded DNA in bacteria
Replicate independently
Can be transferred to other bacteria
Often confer antibiotic resistance.
How can you clone a human gene?
Isolate it and insert it into a plasmid that codes for antibiotic resistance = recombinant DNA molecule
Introduce it into a bacterium
Allow bacteria to replicate
Select for cells containing the plasmid by treating with antibiotic
Why clone human genes?l
Produce proteins like insulin
Find out what genes do
Genetic screening
How is the gene for insulin found?
Isolate mRNA for proinsulin
Use reverse transcriptase to synthesise cDNA (of proinsulin)
Can then introduce into plasmid, then E.Coli
Where is Taq polymerase found?
Thermus aquaticus (lives in hot springs - 90 degrees Celsius, therefore stable at high temperatures)
What primers are required for PCR?
Forward and reverse primers, complementary to 3’ end of each strand of the section of DNA you want to clone.
What is the cycle of temperatures in PCR?
95 - denature
50 - primers anneal
72 - polymerise
Why use PCR?
Amplify a small fragment of DNA
Investigate single base mutations, small deletions and insertions, investigate variation and genetic relationships.
How can proteins be separated?
On the basis of size, shape or charge.
What is protein gel electrophoresis?
Separation of proteins, in their native folded state, in a porous gel with a charge gradient across it, according to their size, shape and charge.
What is SDS-PAGE?
Sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Denature proteins, and is negatively charged so they are separated on size alone (add beta mercaptoethanol to denature disulphide bonds)
Then can do gel electrophoresis- separates on size alone, not charge or shape.
What is isoelectric focusing?
Separation of proteins by charge.
Create a ph gradient. Add proteins. They migrate until they reach their isoelectric point, then don’t migrate any further as they have no net charge.
What is 2D PAGE?
Separate on basis of isoelectric point (pH) then electrophoresis- size
Useful for complex mixtures.
What can 2D page be used for?
Diagnosis of cancers if proteins are upregulated
What is proteomics?
Analysis of all proteins expressed from a genome
Identify proteins by digesting with trypsin, performing mass spectrometry and generating a list of peptide sizes, comparing with a database and hence identifying the protein.
What are the different ways of cleaving a protein?
Enzymes
Chemical cleavage
What is an epitope?
A sequence of a few amino acids on a protein recognised by an antibody
WHat are polyclonal antibodies?
Specific to one antigen, but multiple epitopes - many different B lymphocytes producing many different antibodies
Produced by introducing the antigens into a mouse, then extracting the blood.
What are monoclonal antibodies?
One antibody produced by one B lymphocyte - one epitope, one antigen
What is western blotting?
Electrophoresis of proteins
Transfer to nitrocellulose
Add primary antibodies
Then enzyme linked secondary antibody added - produces luminescence / stain in proportion to the amount of protein.
What is ELISA?
Enzyme linked immunoabsorbent assay
Antibodies fixed to a well.
Solution containing antigens we wish to determine the concentration of is added
Specific antibody binds antigens
Enzyme linked antibody binds specific antibody
Add substrate - converted by enzyme to coloured product - rate is proportional to concentration of antigen.
What can ELISA be used for?
Determine concentration of hormones
What can be used to measure the rate of product formation in an enzyme assay?
Spectrophotometry
Chemoluminescence
Or discontinuous assays - radioactivity or chromatography.
What enzymes are markers for liver damage?
ALT, AST, gamma glutamyl transferase
What enzymes are markers for pancreatitis?
Amylase and lipase
What enzymes are markers for bone disorders?
Alkaline phosphatase
What types of Creatine kinase are present in each of the brain, skeletal muscle and myocardium?
CK1 - BB - brain
CK2 - BM - myocardium
CK3 - MM - skeletal muscle and myocardium
What is the gold standard of diagnosis in MI?
Measurement of cardiac troponin 1 by ELISA.
How is blood glucose concentration measured?
Glucose oxidase catalyses reaction where glucose is the substrate - releases hydrogen peroxide
Test strips are used, hydrogen peroxide is converted to coloured dye.
What are the ethical implications of genome sequencing?
Who owns the information?
Does it have the potential to lead to discrimination (eg insurance)
Can it help prevent illness in later life
Who would be interested in the info - family, potential spouse, police, government, schools, doctors.
What is reverse transcriptase PCR?
Start with mature mRNA (no introns)
Use reverse transcriptase to produce cDNA (use sequence of thy mines as primer - complementary to polyA tail)
Do PCR on DNA.
What can microarray be used for?
Comparison of gene expression by comparing mRNA - tumour tissue
Comparison of genome - insertions/deletions of genes
How do you perform a microarray?
Isolate DNA or RNA Label each with a different probe Combine equal amounts and add to microarray. Allow to hybridise, wash Compare the colour
What does DNA fingerprinting do?
Analyse copy number variation in minisatellites/ small tandem repeats
What does DNA fingerprinting show?
Family relationships
Use in paternity disputes and forensics
How are fragments separated in DNA profiling?
PCR, then Southern blot
What is karyotyping?
Stain chromosomes to show banding
Sort them to check chromosome number, large insertions, deletions or rearrangements
What is FISH?
Fluorescence in situ hybridisation - denature chromosomes that are fixed to a support.
Add fluorescently labelled probe
Locate a gene on a chromosome.
What is chromosome painting?
Use fluorescent probes that are specific to each chromosome - can identify rearrangements in tumours etc
