Recombinant Technology Flashcards
What is recombinant technology?
Combining DNA from diff sources
Involves synthesising
Amplifying & purifying DNA molecules
2 basic principles involved in re DNA?
Research →
1.) → complimentary single stranded nuclei acids will hybridise
Eq.) mixture of strands, single strand → complementarity
( completely diff but comp elementary bases )
Thus able to combine
2.) specific DNA sequence are specific docking sites for specific DNA binding proteins
Define restriction enzyme?
Locate specific palindromic sequence of DNA which they bind to and cut
They cut between 2 specific bases in sequence
Cut 2 strands at diff points leaving sticky ends
Overhang → when pulled apart
What are palindromic sequences?
Read the same 5’ to 3’ on one strand as they do to 5’ to 3’ on antiparrallel strands
How does cutting and ligating DNA fragments work?
Sticky ends can renybridise → from 2 diff sources
This can allow joining of DNA fragments from diff sources as long as they were produced by same enzyme
What is plasmid?
Common in bacteria
Circular DNA molecule that can be replicated within a bacterial cell independently of chromosomal DNA
Was origin of replication and restriction enzyme site ‘
How is DNA inserted into bacterial ‘plasmid’?
1.) The plasmid and the foreign DNA are cut by the same restriction enzyme
2.) when mixed, the sticky ends anneal joining the foreign DNA and plasmid
3.). Nicks in the sugar -phosphate bonds are sealed by DNA ligase
Recumbent plasmid
2 copies occasionally.
How does recombinant plasmid used to ‘transform’ bacterial cells?
Recombinant plasmid canbe inserted back into bacterial cell
As cells replicate, the no of copies of the fragment is “amplified “ as long as it has an origin of replication
Purified recombinant DNA prods
What is polymerase chain reaction? ( pcr)
Allow a specific fragment of DNA to be isolated from a genome
Prods many. Copies of DNA fragment→ PCR amplification
Uses DNA polymerase → synthesise copies of fragments sufficient amounts
Design 2 short DNA’ primers’ → 20 base pairs
Complementary to regions at either end of sequences
2 strands of target DNA are separated
Primers bind to begin synthesis of new DNA strands - s’-3’
The PCR starting point?
DNA is made a single strand by heating to 90°C (breaks hydrogen bondies)
Cooling to 50-60°C allows primers to anneal
Heating to 72°C allows DNA polymerase to synthesise new DNA strands starting from primers
What type of process is per?
Cyclical process: steps 1,2,3 repeated many times
Heating → 90°C
Cooling to 50 - 60°C
Heatin g to 72°C
Amount of your fragments double in each cycle
What DNA polymerase used per?
TAQ polyemorase as it doesn’t denature at ↑ temp (90°C)
How is the PCR product inserted into a bacterial “plasmid” ?
1.) the plasmid and foreign DNA arecut by the same restriction enzyme
2.) when mixed the sticky ends anneal, joining the foreign DNA and plasmid
3.) Nicks in the sugar phosphate are sealed by DNA, ligase
Re combining plasmid can be inserted back into a bacterial cell
Cells replicates → no of copies of fragment “amplified” as long as it has origin of replication
Purified recombinant DNA
Purified recombination cloned DNA?
Makes protein in bacteria
Puts engineered gene into target sequence
Define genetic engineering?
Use of recombinant DNA to alter the genotype, phenotype of another organism
Transgenic = organisms containing foreign DNA
