Week 5 molecular biology Flashcards
Why molecular biology?
Determine what protein cells and tissues contain and in what quantities.
How these proteins are modified and which of these proteins interact with each other.
In short, steps of molecular biology (3)
- produce proteins (recombinant DNA technologies)
in order to be able to look at them:
- separate (SDS-PAGE)
- mark / ways of identifying (western blotting)
Possibilities of recombinant DNA techniques:
Artificially manipulate the protein we’re interested in inside the cell.
Changes to the cell protein (4 variotions)
- force the cells to make extra copies of the protein
- remove the protein that the cell naturally makes.
- to create mutant versions of the protein
- to create tagged versions of the protein
- Ways of effecting phenotype (2)
What info do different phenotypes give?
1.
- level of protein from none to over expressed
- mutant vs. wild type
2.
- what function does the protein have, what processes stops, for example mutating an active part of an enzyme and checking if the enzyme is still catalytically active.
Steps from an interest in a protein to actually having it?
- Interest - need a lot of it
- getting the gene (the actual thing)
that codes the protein (buy or from a colleague)
- Gene comes in a vector, usually plasmid
- researches usually want to change the plasmir to the one they want to use, cloning using PCR, polymerase chain reaction
- makes millions of copies
- bacterial transformation (insert cloned plasmid into bacterial cells)
- bacteria makes thousands of copies
purify plasmid (gene) from bacteria
- now you have LOTS of DNA ready to be used
- expressed in cells (neurons, skin etc.)
- extract protein from cells
vector?
- Is called, and is a piece of what?
- Contains: (2)
A gene needs a ‘place to be in’ - this is called a vector, usually plasmid.
1. - plasmic vector, a circle of DNA found in bacteria
- Contains gene for the protein cDNA
- promoter for the gene
- Contains gene for the protein cDNA
terminology:
Promoter
Isoform
Eukaryotic cells
Prokaryote cells
Introns
nucleotide
Promoter - where transcription of a gene is initiated.
Isoform - different versions of an RNA transcript from the same gene
Eukaryotic cells - cells with nucleus (contains DNA) animals, plants, algae
Prokaryote cells - lacks nucleus (DNA floats in the cell), bacteria
Introns - sections of DNA or RNA that do not code for proteins.
nucleotide - four building blocks of DNA
Four ingredients of PCR - polymerase chain reaction
- template DNA (the DNA we want to amplify)
- DNA polymerase (an enzyme that synthesises DNA)
- primers (short strands of DNA that are complimentary to the start and end of the DNA we want to amplify, shows polymerase which bits to copy
- nucleotides (C, G, A, T)
Explain
Polymerase chain reaction:
- first heating breaks up hydrogen bonds, opens double helix
- cooling, primers bind to the beginnings and end of the target DNA. Shows polymerase where to start the synthesis of DNA
- heated to optimal temperature for the polymerase to start the synthesis. Slides along exposed DNA copies a new strand of DNA
what is used to get the wanted gene into the target plasmid?
PCR, by designing the right primers that will open the plasmid from the right spot (and copied by polymerase).
Linearising plasmic
What is transformation in cloning plasmid?
Bacteria is used to clone the ready plasmid (containing the wanted gene (and antibiotic resistant gene))
Mix of bacteria and plasmid heated to 42’C then cooled rapidly to 4’C.
Thermal shock causes bacteria to take up exogenous DNA (designed plasmid), this is called TRANSFORMATION.
Steps in producing DNA? (
tips:
- $
- PCR
- thermal shock
- after antibiotic-resistant colony has been picked up from agar plate and grown (multiplied) in a culture.
- mutations
- whats the result, goal of the whole damn thing?
- get the gene you want and the plasmid vector you want
- cloning; get the gene from the plasmid it came in into the plasmid you want to use, using polymerase chain reaction PCR
- bacterial transformation, used to produce a lot of the new designed plasmid (contains the gene, and antibiotic resistance gene)
- purify plasmid from bacteria
- sequencing, check the gene for any mutations
- lots of DNA to be used, expressed in neurons, skin cells etc.
Ligation reaction?
In cloning, gene needs to be inserted into the wanted plasmid.
In PCR (before cloning) primers (attaching to the beginning and end of the gene) are designed so that they match the new plasmid one wants to use.
A.
The new plasmid is opened from the right spot, ‘linearised vector’
B.
The gene has matching DNA sequences at the end and beginning
C.
an enzyme is used to separate just a single strand at the ends (of linear vector and the gene). This creates ‘sticky ends’.
D.
The sticky ends bind into a double helix, forms a circle of DNA = complete plasmid with the wanted gene in it!!
sodium dodecyl sulphate-polyacrylamide gel electrophoresis, (shortened)
uses …. to separate proteins in a sample by size - or to put it more accurately, by …. ….. .
SDS - PAGE
uses electrophoresis to separate proteins in a sample by size - or to put it more accurately, by molecular weight.
(SDS - PAGE)
Electrophoresis works by applying an ….. across a gel, in this case, made of a molecule called …….
electric current
poly acrylamide
