Uses of recombinant DNA technology
- production of therapeutic proteins
- investigation of function of genes, enhancer elements, etc.
- generation of novel proteins
- generation of recombinant vectors for gene therapy
- making transgenic plants and animals
How do restriction endonucleases form part of bacterial defense systems
Restriction endonuclease binds to recognition site on invading virus and cleavages both of the DNA strands
- own DNA not targeted as the recognition sites are methylated and bind to methyl transferase
What are the types of restriction enzymes
- Isoschizomers
2. Neoschizomes
What are isoshizomers
Restriction enzymes that recognize the same sequence and cut it in the same way
What are neoschizomers
Restriction enzymes that recognize the same sequence and cute it in different ways (to produce different sticky ends)
Components of plasmid vectors
- circular
- origin of replication
- antibiotic resistance gene + lacZ gene
- MCS
Components of expression vectors
- origin of replication
- antibiotic resistance gene (AmpR)
- T7 promoter
- RBS- ribosome binding site
- T7 terminator
- BamHI site for inserting CDS
Why don’t expression vectors have MCS like plasmid vectors
Because for coding sequence to be read correctly in frame needs to be inserted in specific region
Characteristics of cloning bacteria
- specialized strains of E. Coli
- lack methylation enzymes (so restriction sites aren’t silenced)
- lack recombination ability
- sensitive to specific antibiotics
- partial laxZ in genome
How does directional cloning occur
2 different enzymes produce 2 non compatible ends of the plasmid
What is Sanger sequencing
A method to determine the base nucleotide sequence in DNA
- en corporation of ddNTP which lacks OH group results in no further elongation once included
Sequence of PCR (polymerase chain reaction)
- DNA heated to 90-100 degrees to separate the 2 strands
- The DNA quickly cooled to 30-65 degrees to allow short single stranded primers to anneal their complementary sequences
- Solution heated to 60-70 degrees, DNA polymerase synthesizes new DNA strands, creating 2 new double stranded dna molecules
What type of dna is used in PCR
Archaea dna used as it can withstand without denaturing like E. coli.
Disadvantage is it doesn’t have proofreading properties and so other bacterial enzymes are used in combination to proofread
Steps of RT-PCR (reverse transcription PCR)
- RNA gets converted into DNA by reverse transcriptase and DNA polymerase
- RNA gets degraded by RNase
- Normal PCR of DNA
