Flashcards in Molecular Techniques Deck (64)
What is a restriction enzyme?
An endonuclease produced by certain bacteria that cuts specific DNA sequences (restriction sites)
What do restriction sites usually contain?
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?
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?
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