Molecular Biology (4) Flashcards
Define genetic engineering
Direct manipulation of genome using biotechnology
What are the steps of the polymerase chain reaction (PCR)?
Template DNA - 30s at 94-98°C - denaturation
ssDNA - 30s at 55-70°C - annealing of primer
2 strands with primer each - 2-5mins at 68-72°C - extension
What are the products after 3 cycles of PCR?
2 correct products
6 longer products
What are the types of cloning vectors and what are their capacities?
Plasmid - 15Kb Phage - 25Kb Cosmid - 50 Kb Bacterial artificial chromosome (BAC) - 300Kb Yeast artificial chromosome (YAC) - 1Mb
What is a DNA library?
Collection of cloned DNA fragments
What is a genomic library?
Collection of DNA fragments representing entire genome of organism
What is a cDNA library?
Collection of complementary DNA molecules synthesised from mRNA molecules in a cell - only exons
What are the types of transfection into expression vectors?
Transient
Stable
What are the steps of transient transfection?
Insert ds cDNA for desired protein into plasmid
Transfect cells by lipid treatment/electroporation
What are the steps of stable transfection?
Insert ds cDNA for desired protein into neoR plasmid
Transfect cells by lipid treatment/electroporation
Select for neomycin resistance
Neomycin-resistant clones have cDNA integrated into host chromosome
What is the aim of zinc finger nucleases and how do they work?
Targeted genome modification
Proteins linked to nuclease - also link to base triplets - recognise specific sequence - cleave at this site
What is the aim of transcription activator-like effector nucleases (TALEN) and how do they work?
Targeted genome modification
TALE subunits each bind to one nucleotide - recognise specific sequence - linked to nuclease - cleave at target site
What are CRISPRs?
Clustered regularly interspaced short palindromic repeats
What is Cas9?
Nuclease associated with CRISPR
What is PAM?
DNA sequence where Cas9 cuts
