Test 1 Flashcards
Epigenetic
Changes in DNA reading from behaviour or environmental impact
Herceptin
Drug that treats the HER2 mutation in cancer cells by blocking some of the upregulated transporters
Steps in Molecular Cloning
- Choice of host organism and cloning vector
- Preparation of vector DNA
- Preparation of insert DNA for cloning
- Creation of rDNA with DNA ligase
- Introduction of rDNA into host organism
- Selection of transformants
- Screening of clones
Most common host organism for cloning
E. coli:
Versatile, widely available, easy to grow, minimal equipment
- Not suitable for very large DNA inserts
Common Features of Cloning Vectors
Small Size
Allows replication independent of host chrDNA
Multiple Cloning Site (Polylinker)
Selectable Marker Genes (Selection and propagation)
Primer sequences
Selectable Marker Genes in Plasmid Cloning Vectors
e.g. antibiotic resistance
- Two functions: selection & propagation
- Blue/white
Primer Sequences for DNA Sequencing in Plasmid Cloning Vectors
Commonly bracket the MCS
Facilitate sequencing of cloned DNA inserts
RNA polymerase promoter sequence
In expression vector
Allows expression of RNA and protein
Function of the Regulator/Operator
Roadblock to the Promoter, prevents Gene from being express (e.g. useful with toxic genes)
Typically ‘lac operator’, falls off when exposed to lactose
What sits in the Promoter region
Polymerase
Transcriptional Terminator
Ends the transcription after the inserted gene, stops the Poromoter from continuing past.
Preparation of Vector DNA for Cloning
DNA needs to be isolated and purified
Vector is cleaved by a restriction endonuclease
Preparation of Insert DNA for Cloning
Restriction digest
PCR of genomic DNA
PCR of cDNA
Preparation of synthetic gene from DNA synthesis
Preparation of DNA for Cloning
Purified DNA is treated with compatible restriction endonuclease
Creations of DNA with DNA Ligase
Vector & Insert DNA’s mixed
DNA ligase added to join |DNA ends
Introduction of rDNA into Host Organism
Transformation - Prokaryotic
Transduction
Transfection - Eukaryotic
Electroporation
Selection of Transformants
Presence of selectable markers in vectors ensures only transformants survive
Typically an antibiotic resistance gene
