Lecture 30 - DNA cloning and experimental gene expression Flashcards
Goal of DNA cloning
produce large quantities of identical DNA
General scheme of DNA cloning
Vector + DNA fragment (called insert) give recombinant DNA (any DNA from diff. sources) -> replicated within host cells and manipulated
What is a plasmid + length without essential sequences
Small circles of DNA in prokaryotes, indepedent of big circular chromosomes. w/ essential seq : 1.2 - 3kb
3 important regions on plasmid
Origin or replication, Selectable marker, Polylinker
What is a polylinker
Region with multiple restriction sites (or ‘‘multiple cloning site’’) where insert added
What is a selectable marker
An antibiotic resistance gene (like for ampicillin or kanamycin)
Steps of DNA fragment insertion (3)
Cut vector with restriction fragment, insert the DNA fragment, DNA ligase seals DNA:DNA joints
What is common to all restriction sites and definition of that
palindromic sequence. Same sequence from 3’->5’ on both strands
Most known restriction enzyme and restriction site sequence
EcoRI. Restriction site sequence is (from 5’ to 3’) GAATTC
2 ways of cutting a restriction site + example for each
Leave sticky ends (EcoRI) or blunt cut (SmaI)
Usual sequence length for a restriction site and frequency of 6 bp sites in the genome
4-6 bps, restriction site not hard to find in genome -> one 6 bp sequence every 2-3 kb
Condition for proper insertion of insert in restriction site and ex. w/ EcoRI
Must have complementary sticky end. If EcoRI has cut, need AATT (the sticky end sequence from 5’ to 3’)
Which DNA ligase is common for ligation part and how it uses energy
T4 DNA ligase. Will convert 2 ATP molecules to 2 AMP +PPi
What transformation or transfection means
Putting recombinant DNA in the bacteria
What is cloning ultimately
Being successful in putting one recombinant DNA molecule in a cell
why cloning is difficult
Difficult cause cells don’t like taking up plasmid
Secret to cloning (solution to the difficulty)
Use very dilute recombinant DNA (small number of molecules) so that just a few cells take up one and it is impossible for a cell to get 2
Where bacteria are cultured and characteristic of milieu + why
nutrient agar plates. contain ampicillin to kill bacteria that didn’t take up plasmid
After many colonies of bacteria containing the plasmid are obtained, how is plasmid purified
One clone is picked, put in liquid culture and then plasmid DNA is purified from bacteria
Goal of a DNA library + something particular about genes in a DNA library
Produce many copies of all genes. Genes in DNA library only have their exons
Technique similar to cDNA libraries (but that doesn’t allow multiplication of many genes)
RT-PCR
Step 1 of making DNA library
Get an mRNA mixture from cell extract and produce a cDNA mixture using reverse transcriptase
Step 2 of making DNA library (after we have cDNA)
Make dsDNA out of each cDNA and each dsDNA is an insert that will be put in recombinant DNA (plasmid)
Step 3 of making DNA library (after plasmids done)
Transform bacteria with plasmids containing our cDNA and grow colonies
Step 4 of making DNA library (after we grew colonies)
Each colony contains cells with one of our cDNA, pick colony and isolate plasmid
2 broad reasons for expressing foreign proteins in bacteria or in eukaryotic cells
Biotechnology and Research
Biotechnology : Why express foreign proteins in bacteria or in eukaryotic cells
Produce large amounts of a protein that is rare in nature or difficult to purify or expensive to purify or ethical issues for human sources
Research : Why express foreign proteins in bacteria or in eukaryotic cells
Express wild-type,mutant or fragment of a protein to study function in a cell
What is necessary to drive transcription of the cDNA insert now in a plasmid
Need a promoter
Restrictions about promoters used for expressing cDNA in a plasmid
Must use prokaryotic promoter if in a prokaryote
Eukaryotic promotor of the specific cell type if in a eukaryote
Common prokaryotic promoter + it is required for transcription of WHAT
LacZ promoter. For transcription of an operon of 3 genes, including LacZ gene
What lacZ gene (part of the operon) codes for + function and how LacZ promoter is regulated
LacZ gene codes for beta-galactosidase, prot for lactose metabolism. LacZ promoter regulated by lactose qt.
In transfected bacteria, what substance is used to regulate lacZ promoter expression
IPTG, a lactose analogue
When inserting our cDNA in a plasmid containing LacZ promoter, what do we have to do (we want to express OUR protein)
Remove LacZ gene
2 types of transfection in eukaryotic cells/organisms
1) Transient transfection
2) Stable transfection
What happens to plasmid in transient transfection
Goes in nucleus, many cells take it up but it can’t replicate fast enough
Transient transfection : What happens w/ time to level of protein (of our cDNA) expression
Less cells have the plasmid so less and less protein expression
How eukaryotic cells in culture are transfected (2 methods)
Lipid treatment
Electroporation
In eukaryotic transfection, what selectable marker is used and what drug is put on the plate to kill cells without plasmid (STABLE TRANSFECTION)
Neomycin resistance gene (cause neomycin kills eukaryotes). G-418 put on plate (a neomycin analogue)
Obtaining transgenic mice : Step 1
Inject foreign DNA in one of the pronuclei (2 nuclei of zygote - 2 fused gametes) and transfer injected eggS into foster mother
How DNA injected inserts in pronuclei DNA
High-change of random insertion by non homologous end joining
Effect of random insertion of foreign DNA in pronuclei DNA on genes already there
Most random insertions don’t disturb other genes, but SOME MAY
Obtaining transgenic mice : Step 2 (what happens after injected eggs are in foster mother)
10-30% of offspring contain foreign DNA in chromosomes of all their tissues AND GERM LINE
Obtaining transgenic mice : Step 3 (we have offspring with the foreign DNA in chromosomes of tissues and germ line, what next)
Breed mice expressing foreign DNA to propagate DNA in germ line
Difference between transient and stable transfection
Stable transfection uses selectable marker so only cells that have and express our cDNA survive
