Molecular Techniques Flashcards
What are restriction enzymes?
Endonucleases produced by bacteria
What are the functions of restriction enzymes?
Recognition and degradation of foreign DNA
Recognise and cut specific DNA sequences (restriction sites)
How do restriction enzymes cut DNA?
Break phosphodiester bonds between adjacent nucleotides - giving sticky ends
What is common in the sequences that restriction enzymes usually cut?
They usually cut palindromic sequences
What technique is used to separate DNA fragments created by restriction enzymes?
DNA gel electrophoresis
On what basis does DNA gel electrophoresis separate DNA fragments?
On the basis of size
What is required for DNA gel electrophoresis?
Agarose Gel - matrix that allows the separation of fragments
Buffer - allows charge on the DNA samples
Power Supply - generates charge difference across gel
Stain/Detection - to identify presence of separated DNA (UV)
Where do the DNA fragments in gel electrophoresis start? Why?
At the cathode (-ve electrode)
DNA is negatively charged due to phosphate groups moves towards the positive electrode (anode)
How are DNA fragments separated in DNA gel electrophoresis?
Larger fragments move less far in the gel
Smaller fragments move further in the gel
What can restriction analysis be used to investigate?
To investigate small deletions (size of DNA fragment)
To investigate mutations
To investigate DNA variation
To clone DNA
What vector is used in gene cloning?
Plasmid
What are some features of plasmids?
Small circular DNA found in bacteria
Can transfer to other bacteria (e.g. Antibiotic resistance genes)
What are the steps involved in gene cloning?
Isolate the DNA you want using restriction enzymes
Place DNA into plasmid using DNA ligase (producing recombinant DNA)
Introduce into suitable host cell (e.g. E. coli)
Identify and isolate the clone that contains the certain gene
What is a common application for gene cloning?
Making proteins (e.g. Insulin)
Are copies of genes initially taken in the RNA or DNA form? Why?
RNA form - doesn’t contain introns —> direct code for protein
Reverse transcriptase —> RNA —> DNA —> plasmid
What is PCR useful for?
Amplification of a target DNA sequence
What three things does PCR require?
A thermostable DNA polymerase (Taq polymerase)
Specific forward and reverse primers
Temperature cycling - denature, anneal, polymerise
What is PCR useful for investigating?
Single base mutations
Small deletions
Genetic variation
What are the 3 steps involved in PCR and their temperatures?
Denaturing (95 degrees Celsius)
Annealing (55 degrees Celsius)
Polymerisation (72 degrees Celsius)
What is the function of protein gel electrophoresis?
To separate proteins based on size, charge and shape (usually size)
What 4 things are required for protein gel electrophoresis?
Gel
Buffer
Power supply
Stain
How does protein gel electrophoresis work?
Contents of cell (mixture of macromolecules) placed at wells near cathode in a vertical gel
Larger proteins move less far in the gel
Smaller proteins move further in the gel
When is SDS-PAGE used?
When you want to ignore the intrinsic properties of a protein and separate by size only
How does SDS-PAGE work?
Protein unravelled —> using detergent; SDS and beta-ME
Then separated on a gel using protein gel electrophoresis
What is isoelectric focusing used for?
To separate proteins based on their isoelectric point (charge)
What is the isoelectric point of a protein?
The pH at which the protein carries no charge
How is isoelectric focusing carried out?
Ph gradient created in a gel with an electric field
Proteins migrate till they reach the pH that equals their pI
What is 2D-PAGE used for?
Used to separate complex mixtures of proteins based on charge and size
Important in diagnosing disease states
How is 2D-PAGE carried out?
Usually isoelectric focusing followed by SDS-PAGE
How can proteases be useful in identifying mutations?
Specific proteases cut proteins at certain amino acid residues —> so will change the size of fragments produced
What is an epitope?
Part of the antigen that the antibody binds to
How do antibodies bind to an antigen?
Antibodies recognise a specific amino acid sequence on an antigen called the epitope
What are two types of antibodies?
Polyclonal antibodies
Monoclonal antibodies
What is the difference between polyclonal and monoclonal antibodies?
Polyclonal antibodies recognise multiple epitopes on a specific antigen
Monoclonal antibodies recognise 1 epitope on a specific antigen
What is western blotting used for?
Using antibodies to detect a protein
How does western blotting work?
SDS-PAGE used –> addition of primary antibody which binds to the protein –> addition and binding of radioactively labelled secondary antibody —> detection of protein
What is ELISA used to measure?
Measures the concentration of proteins in a solution
How does ELISA work?
Similar to western blotting, enzyme linked antibody binds to the protein –> substrate added and rate of reaction measured which is proportional to concentration
What is radioimmunoassay?
Used to measure the concentration of a product
Uses radiolabelled primary antibody
What are enzyme assays used for?
To measure the activity of an enzyme in solution through measuring of a product
What are two methods of continuous assays?
Spectrophotometry
Chemoluminescence
What are two methods of discontinuous assays?
Radioactivity
Chromatography
What can be used to measure the concentration of metabolites?
Enzymes
What is dsDNA and ssDNA?
Double stranded DNA
Single stranded DNA
How can dsDNA be used to form ssDNA?
Denaturing - breaking of hydrogen bonds with heat or a pH>7
How can dsDNA be formed from ssDNA?
Renaturing - hydrogen bonds reformed
Cooling or pH=7
How can a DNA probe be used?
Denaturing of dsDNA into ssDNA —> addition of complementary ssDNA sequence which is radioactively labelled —> identification using photographic film
What are two hybridisation techniques?
Southern blotting
Northern blotting
What is southern blotting?
Uses DNA probes to identify complementary sequences after gel electrophoresis
What is northern blotting?
Similar to southern blotting, where DNA is used to detect RNA species
Is western blotting a hybridisation technique?
No - is the detection of proteins by antibodies after protein gel electrophoresis or SDS-PAGE
What is the process of southern blotting?
Digest DNA with restriction enzymes Separate with DNA gel electrophoresis Transfer dna fragments to nylon or nitrocellulose to denature Add radioactively labelled DNA probe Detection using X-ray film
Why is southern blotting used?
Used to detect dna fragments from complex mixtures
What are some characteristics of DNA probes?
DNA probes do not have to be 100% identical to the target sequence - will stand bind but less tightly
DNA probes do not have to completely align with the target sequence
By which method is DNA sequencing carried out?
Sanger Chain Termination Method
What is a ddNTP?
Dideoxynucleotide triphosphate
Same as a normal deoxynucleotide triphosphate —> but has a H atom on the 3rd carbon rather than a hydroxyl group
How are ddNTPs incorporated into a growing DNA strand?
Can be incorporated by DNA polymerase but will prevent further elongation of the chain as no further phosphodiester bonds can be formed
How is sanger chain DNA sequencing carried out?
Labelled primer + all the dNTPs + 1 ddNTP + DNA polymerase all placed in a test tube
Different length DNA fragments produced depending on where ddNTP incorporated
4 separate test tubes used, DNA fragments separated by size on a gel
Gel is read from the bottom to work out the nucleotide sequence
What techniques are used to investigate DNA at a chromosome level?
Karyotyping
FISH
Chromosome Painting
What techniques are used to investigate DNA at a nucleotide level?
DNA sequencing
PCR + restriction analysis
What techniques are used to investigate DNA at a gene level?
Southern hybridisation Northern hybridisation RT-PCR Microarray DNA fingerprinting
How can PCR be used to identify mutations?
By using allele specific primers
How does northern hybridisation differ to southern hybridisation?
Uses DNA probes to look at RNA species and investigate gene expression rather than gene structure
What is RT-PCR used for?
Used to look at gene expression
How does RT-PCR work?
A primer is added with a long stretch of T’s at the 5’ end to a mature mRNA molecule with a polyA tail
Complementary sequence extended using reverse transcriptase
RNA broken down with RNAases
Forward and reverse primers added –> normal PCR reaction
What is microarray used to investigate?
To compare gene expression between two different sets of genes
Allows the analysis of 1000s genes at the same time
What is DNA fingerprinting used to investigate?
To look for family relationships
How is dna fingerprinting used to look for family relationships?
There are repeating sequences on the non-coding part of DNA called minisatellites - these are unique to each individual and are inherited from mother and father –> so no individual will have the same
Similarities between these sequences can be used to prove family relation
What is karyotyping? How can it be used to analyse chromosomes?
The extraction and staining of chromosomes at the metaphase stage
Specific sizes/banding allows sorting and comparison
What is FISH? How is it used?
Fluorescent in situ hybridisation - happens inside the cell
DNA probe with fluorescent dye added to cell
Denaturing and hybridisation of DNA
Detection of dna probe and its position on the chromosome take place
