Molecular Biology 4 Flashcards
What is genetic engineering ?
Direct manipulation of the genome of an organism using biotechnology.
What are the three steps of PCR ?
How long does each of them last ?
At what temperature is each performed ?
Denaturation : 30s, 94-98 degrees
Annealing : 30s, 55-70 degrees
Extension : 2-5min, 68-72 degrees
After which PCR cycle do we obtain the 2 copies of our fragment without any extensions ?
Cycle 3
What does PCR allow ?
Exponential increase in the amount of a specific sequence of DNA (followed by a linear increase and a plateau).
What is another name for the Sanger sequencing method ?
The di-deoxy chain terminator method.
Later became the dye terminator method.
How does NextGen sequencing compare to the Sanger method ?
NextGen Sequencing can read >3GB of DNA /per run/day (thus whole haploid human genome)
The Sanger method needs > 2 years to read the same amount of DNA
What are the steps of NextGen sequencing ?
- cut genomic DNA
- add linker
- attach fragments of DNA to flow cell
- bind the fragments to primers
- perform in situ PCR
- dissociate fragments from primers
- sequencing
What is the size (DNA cloning limit) of : - plasmids ? - phages ? - cosmids ? - BACs ? - YACs ? What are each used for ?
Plasmid : up to ~15Kb --> shuttle Phage : up to ~25Kb --> protein expression Cosmid : up to ~50Kb --> reporter gene BACs : up to 300K YACSs : > 1 Mb
What are the main properties of plasmids ?
Found in bacteria
Extrachromosomal, circular DNA
Often contain genes for resistance to antibiotics or production of toxins
Replicates independently of bacterial DNA Replicates autonomously
Has been modified for use in DNA manipulation
How can we generate DNA recombinant molecules with plasmids ?
- take plamids vector (w/ ampR) and the DNA fragment to be cloned
- enzymatically insert DNA into plasmid vector
- mix E Coli w/ recombinant plasmids in presence of CaCl2 (to make E Coli competent); heat pulse
- culture on nutrient agar plates conataining ampicilin
- ONLY transformed cells survive
- cell multiplication (v rapid replication ~every 20mins => exponential growth, 1 bacterium can replicate into 5E21 in one day)
- obtain a colony of cells, each containing copies of the same recombinant plasmid
What is a DNA library ?
A genomic library ?
A cDNA library ?
- a ‘DNA library’ is a collection of cloned DNA fragments
- a genomic library contains DNA fragments representing the entire genome of an organisms
- a cDNA library contains only complementary DNA molecules synthesised from mRNA molecules in a cell
What is the main difference between genomic DNA clones and cDNA clones in eukaryotes ?
Genomic DNA library = contains some introns + some nontrancribed DNA
cDNA library = synthesized from mRNA, no “junk”
How can colonies be screened by hybridization ?
- take a master plate of E Coli colonies
- place nitrocellulose filter on plate to pick up cells from each colonies
- incubate filter in alkaline solution to lyse cells and denature released plasmid DNA
- hybridized probe
- perform auto-radiography (signal appears over plasmid DNA that is complementary to probe)
OR - incubate w/ labelled DNA
- wash away labeled DNA that does not hybridize too DNA bound to filter
- perform auto-radiography
What are the 2 types of transfections that can be performed ?
Describe each of them.
Transient transfection :
- transfect cultured cell by lipid treatment or electroporation w/ a vector containing a viral origin of replication
- protein is expressed from cDNA in plasmid DNA
Stable transfection
- transfect cultured cell (like above) w/ a vector containing for exemple the nerR gene
- select for G-418 resistance
- protein is expressed from cDNA integrated into host chromosome
How can GFP technology be exploited ?
We can add the GFP gene sequence right behind the cDNA sequence (itself behind the promoter) to get the following sequence : promoter -cDNA - GFP
The cDNA-GFP rpresent our reporter construct
