Molecular Techniques Flashcards
What is a restriction enzyme?
An endonuclease produced by certain bacteria that cuts specific DNA sequences (restriction sites)
What do restriction sites usually contain?
Palindromes
What is a sticky end?
Where an endonuclease produces a staggered cut, leaving a few unpaired DNA nucleotides of one strand that extend beyond the other
What attribute does DNA gel electrophoresis separate fragments of DNA based on?
The size of the DNA fragment
In gel electrophoresis, what do DNA fragments move towards the positive or negative electrode? Why?
DNA fragments move towards the positive electrode, as they are negatively charged - DNA is negatively charged due to its phosphate groups
How will a small fragment of DNA move in comparison to a large fragment of DNA in gel electrophoresis?
A small fragment will move further, closer to the positive electrode, in comparison to a larger fragment
What is a plasmid? What specifically may a plasmid contain relevant to medicine?
A small circular double-stranded form of DNA found in bacteria - specifically, they may contain genes that infer antibiotic resistance
Describe the 4 basic steps of gene cloning.
- isolate the relevant gene of interest using restriction endonucleases
- insert this gene of interest into a plasmid vector, producing a recombinant DNA molecule
- introduce this recombinant DNA to a suitable host cell
- identify and clone the host cell containing the DNA of interest
Why would we clone human genes?
- to produce a protein of interest on a mass level
- find out what genes do
- screen for certain disorders (genetic screening)
What holds complemtary nuclear acids in a DNA sequence together?
Hydrogen bonds
Why is a primer needed in PCR?
Taq polymerase can only extend a double-stranded DNA sequence, so a primer must be used to provide the double-strand
In PCR, what temperature is the DNA first heated to? Why is this?
The temperature is raised to around 95C - this breaks the hydrogen bonds that hold the 2 complementary strands of DNA together
In PCR, once the double strands have been separated, what happens next? What does this allow?
The temperature is cooled to room temperature - this allows the primers (needed for Taq polymerase to begin double-strand synthesis) to anneal to the single stranded DNA sequences
What is special about Taq polymerase?
It is able to withstand extremely high temperatures (such as this that are enough to break DNA strands apart) without denaturing
What type of DNA changes is PCR used to investigate?
To investigate small insertions/deletions within a gene
In PCR, are the primers known beforehand?
Yes, in order to amplify the specific sequence that you want to mass produce (you therefore must also know the sequence you want to copy)
What 3 attributes can proteins be separated on in protein gel electrophoresis?
- size
- shape
- charge
How does protein gel electrophoresis separate proteins based on their size?
Larger molecules won’t pass through the viscous gel as quickly as smaller molecules will
What is SDS-PAGE short for?
Sodium dodecyl sulphate polyacrylamide electrophoresis
What does sodium dodecyl sulphate (SDS) do?
SDS breaks down disulphide bonds, breaking a proteins secondary and tertiary structure and leaving only its primary structure (i.e. a linear polypeptide chain) - SDS also adds negative charge to this linear polypeptide chain
What does SDS-PAGE separate proteins based on?
It separates different proteins on the basis of their size
What does isoelectric focusing separate proteins based on?
It separates proteins based on their charge
Briefly explain how isoelectric focusing works.
A stable pH gradient is established within a gel after the application of an electric field - different proteins are added, and migrate to the point where they each a pH equal to their isoelectric point - the gel is then stained to show where the proteins are distributed
In isoelectric focusing, why do proteins stop migrating once they have reached the pH where they are at their isoelectric charge?
They have no net charge at their isoelectric point, and so will have no net charge at this pH, so will stop migrating
Briefly describe how 2D-PAGE works.
2D-PAGE firstly separates proteins by their isoelectric point - having done this it then separates proteins based on their size
When is 2D-PAGE particularly useful?
When diagnosing disease states in different tissues
What is proteomics?
The analysis of all proteins expressed in a genome
At which residues does trypsin cleave a protein?
Lysine & arginine
At which residues does chymotrypsin cleave a protein?
Tyrosine, phenylalanine, leucine, & tryptophan
How are polyclonal antibodies produced?
An antigen is first injected into an animal to initiate an immune response - after a series of time the animal should have developed antibodies against the antigen - blood is then extracted from the animal and purified to obtain the antibody
How are monoclonal antibodies produced?
Ask l8r
Describe how a western blot is performed.
Firstly, proteins are separated by their size (usually via SDS-PAGE) - these proteins are then transferred to a nitrocellulose membrane where they are stained with a primary antibody specific to the target protein - the membrane is rinsed to remove unbound primary antibody and secondary antibody is added to the membrane - this binds the primary antibody, and is normally attached to a bioluminescent molecule or enzyme used to amplify the signal
Describe how ELISA is performed.
Antigens from a sample are attached to a surface of a well - a specific primary antibody is added which binds the antigen - a secondary antibody is added to the well which is bioconjugated to an enzyme - the enzymes substrate is added, which will usually produce a marked change in the colour of the solution - the greater the change of colour, the greater the amount of antigen in the initial solution
Overall, what does ELISA show?
ELISA identifies the presence of a particular substance, or the concentrations of the substance (usually protein) in solution
What is the Vo?
The initial rate of reaction
What is a continuous assay? Give 2 examples.
Where only one assay is needed to get a measure of the enzyme activity - examples include:
- spectrophotometry
- chemoluminesence
What is an enzyme assay? Why are they important diagnostically?
A method measuring the activity of an enzyme - they are important diagnostically as they provide a measure of metabolic function of an enzyme
What is a discontinuous assay? Give 2 examples.
Where several assays/samples are taken from an enzymatic reaction at several intervals to measure enzymatic activity - examples include:
- radioactivity
- chromatography
What 2 methods can you use to denature a double-stranded portion of DNA? Specifically, what do these methods do?
By heating them or subjecting them to an alkaline pH - both of these break the hydrogen bonds connecting the double stranded DNA
What does a Southern blot detect?
A southern blot is used to detect complementary DNA sequences (1 strand from the original DNA and 1 radioactive probe)
What does a southern blot show?
The presence or absence of a particular DNA sequence, by using a radioactive probe
In southern blotting, why is a DNA probe used?
After the DNA is cleaved by a restriction enzyme and then run over gel electrophoresis, the quantity of DNA, and the variation in different sizes, means all that is visible is a huge smear - a probe allows you to specifically identify the sequence of DNA you are probing for
Describe the process of a southern blot.
Firstly, DNA fragments are run over an electrophoresis gel (smaller molecules will move further than larger DNA fragments) - the gel is soaked in an alkaline solution which denatures the DNA into single fragments - these are then transferred onto a nylon or nitrocellulose membrane - the filter is placed into a solution containing the labelled DNA probe, which will bind complementary single-stranded DNA on the filter - afterwards the filter is washed to remove any unbound probe and photographic film is used to detect binding
Why is southern blotting used?
In order to analyse a gene for large duplications, repeats, or deletions, and to investigate for mutations or variation
In southern blotting, do probes have to be 100% complementary?
No - even with 80% homology a DNA probe will bind, albeit less tightly
In southern blotting, do probes affect the position of a target sequence on a gel?
No, they have no effect
What is the best molecular technique to use to figure out a DNA nucleotide sequence?
The Sanger chain termination method
What specific molecule allows identification of the DNA sequence in the Sanger chain termination method?
Dideoxynucleotide triphosphate - this has a H at the 3’ position as opposed to a OH- - this means that when inserted into a sequence, synthesis will end prematurely as a phosphodiesterase bond linking the next deoxynucleotide triphosphate in sequence will not be able to be formed, blocking elongation
On a single strand of DNA, how are subsequent deoxynucleotide triphosphate molecules added?
Through the formation of a phosphodiesterase bond between the 5’ phosphate group and the 3’ hydroxyl group on the growing chain
Describe the process of the Sanger method.
4 seperate tubes, each with a unique dideoxynucleotide triphosphate, are incubated with the target sequence - once incubated, these are run on a gel to determine the order of the nucleotide sequence in the DNA strand - in reality, nowadays radioactive dideoxynucleotide triphosphates are used in one tube and ran over a gel - the sequence is then determined by the next radioactive didepxynucleotide triphosphate that has halted elongation
What can a northern blot detect?
How much mRNA is expressed in a certain tissue
What feature of mRNA does reverse transcriptase PCR utilise? How?
The polyA tail - a 5’ primer with abundant thymine will bind the 3’ polyA tail, and reverse transcriptase will then be able to copy the mRNA
What enzyme is used to separate the double-stranded RNA?
An RNAase
What does each well in a microarray consist of?
Known sequences of DNA
What can microarrays give?
A comparison of 2 conditions, based on the difference in colour of the wells of the microarrays
Describe how a microarray works?
Reverse transcriptase PCR is used to make cDNA from mRNA - the cDNA can then undergo PCR and be amplified - from here, cDNA from 2 different conditions (eg wild type and disease-state) can be separately labelled - these can be mixed and hybridised to a microarray - depending which DNA sequences fluoresce or not, and comparing the levels of fluorescence of the colours you can see the difference in the composition of the DNA across the 2 conditions
When Karyotyping a selection of chromosomes, what stage of nuclear division was the cell they were taken from at?
Metaphase
What is Karyotyping?
Stain chromosomes, and pair them using their stains as markers for a homologous pair - this can then be used to compare deletions on a chromosomal level
Describe the stages of DNA fingerprinting.
DNA is first extracted, then amplified using PCR - a southern blot is then performed, running the DNA fragments down an electrophoresis gel and transferring them to a nylon or nitrocellulose filter for hybridisation with a radio labelled probe molecule - this can then be compared to another DNA fingerprint to identify differences (if any) between the 2 DNA fingerprints
In DNA fingerprinting, what specific sequences of DNA tend to be looked at? Why?
Usually short-tandem repeats are analysed - as they reside in non-coding regions (introns) they show great variability between individuals who aren’t related, and less variability between individuals who are closely related
Why may a short-tandem repeat show great variability?
They are apparent in introns and so are not greatly conserved - therefore, mutations may occur here that have no effect on an individual’s viability so can persist - related individuals however tend to have less variation as they have directly received these short tandem repeats from family members
What type of abnormalities does FISH (fluorescence in situ hybridisation) identify?
Chromosomal abnormalities
What does FISH (fluorescence in situ hybridisation) help identify?
It allows you to identify where specifically on a chromosome a gene resides, and whether there are nay abnormalities within this gene across other chromosomes/samples
What stage of replication are chromosomes analysed for FISH (fluorescence in situ hybridisation) taken?
Metaphase (or interphase)
