Introduction to methods in molecular biology Flashcards
What is the aim of recombinant DNA technologies?
To identify and manipulate proteins:
- additional copies of protein
- removed protein (is it essential to a process?)
- mutated protein (what part of it is important for its function?)
- tagged protein (to find a protein of interest)
What is necessary to study a protein?
We need to make it in significant amounts within working cells
-> get a hold of the gene that codes for the protein (buy it or get it from colleague)
What are the steps in investigating a particular protein, using recombinant DNA technologies?
- Get the gene of interest in a plasmid vector
- choose plasmid vector with the needed promoter and tag - Clone the gene using PCR
- clone gene into target plasmid vector - Bacterial transformation
- insert cloned plasmid into bacteria cells
- > thousands of copies - Plasmid purification
- to have a lot of DNA to work with - Expression of protein in cells
- Extraction of proteins from the cells
- use of plasmids
What are the characteristics of a plasmid vector?
> It’s a circular piece of DNA
Gene inside is complementary DNA (cDNA)
May have eukaryotic resistance marker
Holds exact sequence of the gene
No introns or up/downstream sequences
Can only express 1 isoform
-> choose which isoform of the protein you want
What is the use of an antibiotic resistance gene in a plasmid vector?
To selectively grow bacteria containing the specific plasmid vector
- if the plasmid includes an antibiotic-resistance gene, the bacteria will contain it
What is a promoter in a plasmid vector?
Where transcription of a gene is initiated
- it will be activated in the type of cells we want our protein to be expressed in
- > choose it carefully
What is the process of gene transcription?
When a section of DNA is copied to make mRNA
- RNA may then be used as template to make a protein
What is complementary DNA (cDNA)?
DNA that is synthesised by using RNA as a template
- often used to clone eukaryotic genes in prokaryotes
What is a prokaryote?
Unicellular organism that lacks a membrane bound nucleus
- e.g. bacteria
What are eukaryotic cells?
Have a membrane bound nucleus that houses the genetic material
- make up our body
What are introns?
Sections of DNA or RNA that do not code for proteins
What are up/downstream sequences?
> A gene has 2 ends
- 5’ end
- 3’ end
> Upstream: sequences that are towards the 5’ end
Downstream: sequences that are towards the 3’ end
> Up/downstream sequences are involved in the control of transcription
What are gene isoforms?
Different versions of RNA transcripts made from the same gene
What is polymerase chain reaction (PCR)?
Tool for amplifying the gene from original plasmid and cloning it into target plasmid vector
What are the PCR components?
> Template DNA
- DNA we want to amplify
> DNA polymerase
- enzyme that synthesises DNA
> Primers
- short DNA strands complimentary to the start and end of template DNA
> Nucleotides
- the 4 nucleic acid bases that make DNA: A, C, G, T
What are the 3 phases of a PCR cycle for copying a gene?
- DNA denaturation
- Primer annealing
- Primer extension
What happens during DNA denaturation in PCR?
Initial heating to high temperature breaks bonds holding the 2 strands of template DNA
-> DNA double helix splits open
What happens during primer annealing in PCR?
> Denatured DNA is cooled
> Primers bind to the start and end of template DNA sequence we want to amplify
- show the DNA polymerase which length of DNA to copy
What happens during primer extension in PCR?
> Mixture is heated to optimum temperature for DNA polymerase to work on the exposed DNA strand
> DNA polymerase slide along DNA strand and links spaces together
- > synthesises new DNA strand
- > double amount of DNA
How to amplify the gene copied with PCR?
Repeat the cycle of:
- heating (DNA denaturation)
- cooling (primer annealing)
- warming (primer extension)
-> we double the amount of DNA at each cycle
What makes gene amplification using PCR an exponential process?
At each cycle (heating, cooling, warming of DNA) we double the amount of DNA
- template DNA: 2 original strands
- 1st cycle: 4 copies
- 2nd cycle: 8 copies
- 3rd cycle: 16 copies
- 4th cycle: 32 copies
What are the steps to get the gene out of the original plasmid and clone (insert) it into the target plasmid?
- Gene amplification
- make multiple copies of the gene from original plasmid - Linearise and open the target plasmid to insert the gene
- PCR reaction will generate millions of copies of linearised plasmid - Cloning
- insert the gene into target plasmid vector - Ligation reaction to close target plasmid vector
How do we linearise and open the target plasmid to insert the gene?
Design primers that will bind to the point on the plasmid where we want to open it, which will amplify in opposite directions
What do you obtain when the PCR reactions are complete?
- The gene we want to insert with sequences from the target plasmid now added to the end of the gene’s sequence
- A linearised plasmid vector for the amplified gene
to be inserted into
What type of primers do you need for the gene to be cloned (inserted) into the target plasmid vector?
Primers where:
- half of primer will bind to the gene we want to amplify
- other half of primer is a copy of the part of the target plasmid where we’ll inject the gene
- > primers are incorporated into target plasmid
- > copy of the gene with same DNA sequence as the target plasmid (where we want to insert it into)
How do you clone (insert) the gene into the target plasmid vector with PCR?
We use an enzyme to remove base pairs from the ends of one DNA strand
-> create single-stranded lengths of DNA at the ends of the linearised target plasmid AND the ends of the gene to be inserted
-> the ends of gene PCR products are same as target plasmid
=> Complimentary sticky ends that will line up by complimentary base-pairing = inserting gene into target plasmid vector
How do you close the target plasmid vector where the gene has been cloned into?
Ligation reaction:
- use DNA ligase to close the phosphodiester bonds
- > connects the inserted gene to the target plasmid
=> creates a complete plasmid vector that can be used to transform the bacteria
What are the steps to produce the cloned plasmid vector in usable quantities?
- Transform a bacteria with the cloned plasmid
- We grow the bacteria in a culture
- bacteria replicates the plasmid for us - We can then purify the plasmid from the bacterial cells
- DNA sequencing to confirm the gene has been correctly inserted in the plasmid vector
- We’re ready to use the plasmid in experiments to express the protein of interest in living cells
What is the process to transform and grow the bacteria with the cloned plasmid?
- Plasmid is mixed in with the bacteria
- We heat them to 42° C for a few seconds then immediately cool them to 4° in a box of ice
- > thermal shock causes cells to take up the plasmid from their surroundings - Bacteria take up exogenous DNA = transformation
- We spread the bacteria on an agar plate and incubate them over night
- Following day, a colony can be picked off the plate and grown in a culture
How do we ensure that we obtain only bacteria containing the plasmid?
> If the plasmid included an antibiotic-resistance gene, the bacteria will contain it
> We can then grow the bacteria on an agar plate containing the antibiotic
=> selective growth of bacteria containing plasmid vector
Why is it important to DNA sequence the whole insert of plasmid vectors?
In case any unexpected mutations were introduced during the PCR
What is SDS-PAGE?
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis:
> Separates proteins in a sample by molecular weight
- lower weight molecules migrate across the gel quickly
- higher weight molecules migrate more slowly
> Works by applying an electric field across a polyacrylamide gel
What are the steps of SDS-PAGE?
- Sample preparation
- break down tissue sample
- denature the proteins
- add electric charge to proteins - SDS-PAGE
- separate protein molecules using electric field - Protein preparation
- switching the field off at right time to spread the proteins evenly and separate them
How is the tissue broken down for SDS-PAGE?
> Mechanical breakdown:
- uses homogeniser or ultrasonic vibration
- tears tissues apart, preserving some cell structure
> Chemical breakdown
- mixing cell with a lysis buffer to break the membranes apart
=> cell homogenate or cell lysate
In the sample preparation for SDS-PAGE, why do we need to process the proteins further than their breakdown?
- Proteins won’t move through the polyacrylamide gel on their own
- Different proteins = different shapes
- > could confuse results
=> Any differences in migration during SDS-PAGE should be based only on molecular weight of the protein molecules
During sample preparation, how do we process proteins for SDS-PAGE?
> Tissue breakdown -> cell homogenate or cell lysate
> We treat cell lysate with sodium dodecyl sulphate (SDS)
- acts as denaturing agent
- has long hydrophobic tail: binds to protein and pulls its structure -> unfolds the protein
- has negatively charged head: once SDS is bound to protein -> protein has strong negative charge too
- we use this electric force to move the proteins through the gel
> We add blue dye (often brilliant blue), glycerol, and reducing agents (DTT or beta-mercaptoethanol)
What is the role of the dye added to the proteins for SDS-PAGE?
Enables us to see the proteins when moved through the gel
What is the role of the glycerol added to the proteins for SDS-PAGE?
Makes the sample heavy so it sinks into the well when loading the gel
What is the role of the reducing agents added to the proteins for SDS-PAGE?
Break any disulphide bonds in the proteins
What is the SDS-PAGE separation process?
> Cross-linking lots of polyacrylamide molecules to create a gel mesh
- contains pores of different sizes, which helps to separate the proteins as the larger molecules can’t fit through smaller pores
> Prepared samples are pipetted into wells on the gel
How is the polyacrylamide gel made in SDS-PAGE?
> Acrylamide is mixed with SDS containing buffer
> 2 catalysts (ammonium persulfate and TEMED) are added to initiate polymerisation reaction
-> makes acrylamide molecules cross-link to form the gel
What are the steps of the electrophoresis set up and process in SDS-PAGE?
> Proteins in wells are loaded
- when they hit the join between the stacking gel and resolving gel -> changes of pH and acrylamide slows them down
- proteins entering resolving gel first are slowed down
- > allows others to catch up
> Samples get stacked up, forcing them into a tight thin band in resolving gel
> Buffer allows the current to flow through the gel
- contains SDS and denaturing agents
- stabilises pH of the system without reacting to samples or gel
- SDS assures proteins stay denatured throughout the process
> The closing lid completes the electrical circuit
- electrodes connected to power supply
- alter voltage passing through gel to control protein migration speed
(1-3 hours for samples to reach bottom of the gel)
What are the 2 gels in the electrophoresis set up of SDS-PAGE?
What are their characteristics?
- Stacking gel:
- low pH
- low percent acrylamide - Resolving gel:
- separates the proteins
- varying percent acrylamide
- low acrylamide % -> bigger pores, good resolution for large proteins but not smaller proteins
- high acrylamide % -> smaller pores, good res. for small proteins but not for larger proteins
What happens if there is no stacking gel in the electrophoresis set up of SDS-PAGE?
Proteins would all enter the resolving gel at different times
-resulting in large smeared bands
- we need a tight thin band
How are the SDS-PAGE results analysed?
> At the end of the electrophoresis, proteins are spread through the gel according to their molecular weights
> Proteins have been stained with a blue dye
> Standard size markers on the left side of the gel:
- help approximate molecular weights in rows across gel
- contain mix of proteins of known size to compare with proteins of interest
Why do we need the western blotting technique after SDS-PAGE?
There are so many proteins in the sample, we are unable to identify our proteins of interest
What is western blotting?
> Used to detect specific proteins from a mixture of proteins
> During western blotting, we are processing further the polyacrylamide gel
> Proteins from the gel are transferred to a membrane which blots the proteins from the gel
What are the steps of western blotting?
- Transfer of proteins from gel onto membrane
- Blocking the membrane
- Primary antibodies added, bind to protein of interest
- Incubate the membrane with secondary antibodies
- bind to primary antibodies
- have fluorescent tag or HRP enzyme - Adding substrate for HRP
- enzyme catalyses a chemical reaction producing light
- detected with photographic film or imager
How are the proteins from the gel transferred to the membrane used in western blotting?
> ‘sandwich’ of: positive electrode, sponge, filter paper, membrane, gel (with samples), filter paper, sponge, negative electrode
> Membrane is made from nitrocellulose or PVDF
- good protein retaining properties
> Electrical current runs through the ‘sandwich’
- proteins being negatively charged are forced towards positive electrode, moving out of gel and into membrane
> Once out of transfer, only the parts of the membrane adjacent to the proteins in gel will have proteins on them
How is the membrane blocked in western blotting?
> Cover up the parts of the membrane which do not already have protein on
> We use a solution with a lot of proteins
- usually milk, easily available
Why use antibodies in western blotting?
Allow us to see only our proteins of interest
How is the blot analysis conducted in western blotting?
> Computer programs to measure the intensity of the band on the blot
> Within certain range, the intensity is proportional to the amount of protein in sample
What are the advantages of western blotting?
> Relatively rapid, takes 1 and half day
- can be faster
> Does not require a lot of specialist equipment
> Will work for a wide range of proteins
> Can be sensitive and specific with good antibodies
> Can be applied to many cells, tissues, experimental procedures
> Semi-quantitative: size and intensity of band is proportional to amount of proteins
What are the disadvantages/drawbacks of western blotting?
> Only as good as your antibody
> Antibodies are expensive
> Post-translational modifications may alter binding or mobility
> Does not work as well if your proteins are very large or very small
> Reasonably sensitive, but does not work well if protein levels are low
> Semi-quantitative: you can only see approximative changes within a blot
-> no comparison between membranes
What is important to remember regarding artificial systems and molecular biology?
> Artificial systems can be affected by a range of factors
> Important to use a variety of techniques to understand molecular events inside the cell
