13 - recombinant DNA Flashcards
recombinant DNA
Recombine DNA from two distinct sources. (DNA fragment of interest + plasmid vector)
Conclusion of griffiths experiment
A chemical substance from one cell is genetically transforming another cell
restriction enzyme
cut DNA into small fragments, endonucleases, recognise restriction sites, palindromic, make sticky ends and blunt end cuts (staggered or complementary)
Vector
a DNA molecule that replicates within a living cell, eg plasmid or virus. Contain genetic signals for replication, inserting foreign DNA and identifying cells that contain recombinant DNA
Types of vectors
- Plasmid
- Phage lambda (bacteriophage)
- Cosmid
- Bacterial artificial chromosome
Plasmid
Circular dna molecule that replicates seperate from chromosome DNA. Occur naturally in bacteria and are replicated before every cell division
What is a plasmid polylinker
contains recognition sequence for restriction enzymes
DNA ligase
catalyses phosphodiester bond formation, requires ATP
How to get recombinant DNA into cell?
Transformation. The uptake of exogenous DNA through the cell membrane. Bacterial cells that take up plasmid can be selected
Transforming principle experiment
- Virulent S strain bacteria = mouse dies
- Non virulent R strain bacteria = mouse lives
- heating kills virulent s strain bacteria = mouse lives
- mix dead s strain bacteria and living non virulent = mouse dies
Library
Collection of N vectors with different inserted fragments of DNA
3 types of gene libraries
- DNA library
- Genomic library
- cDNA library
Genomic library
Clones that collectively represent all DNA sequences of the genome
DNA Library
Collection of clones containing all the DNA fragments
cDNA library
Clones representing all mRNA in a cell (mRNA cloned as cDNA into vector)
Screening DNA libraries
DNA libraries can be screened by hybridisation to a DNA probe
Uses of recombinant DNA tech
Pharamaceuticals, medical testing, research, GM food, transgenic animals
Glofish
Insertion (recombinant plasmid injected into cytoplasm of 1 or 2 cell stage embryos)
screening (protein expression began) resulting in glowing fish
Vector for glofish
plasmid pEGFP-1
Restriction enzymes for GloFish
EcoR1
Cloning alternative
Use PCR to make copies of DNA fragment instead of E.coli
Genome engineering
Methods that allow changes to be introduced into DNA of many organisms by inducing a targeted double strand break into the genome
