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
Transgene?
Introduced foreign gene?
E.g. ) production of human blood clotting factor VII in bacteria
The coding region of the human factor VII gene can be cloned into a plasmid called on expression vector
Gene is inserted next to a bacterial gene promoter which will attract the normal bacterial RNA polymerase
Genetic engineering in plants ?
Plants con prod ↑ complex proteins that may need additional processing beyond what A bacterium can do
GE in plants uses natane genetic engineer?
?
Define totipotency?
Any single cell can regenerate a new organism
How does regeneration of transformed plants → a property unique to plant collie?
Totipotency
1.) take cells from host plants
2.) infect a single cells with Agra bacterium
3.) select transformed cells
4.) culture
5.) add rooting and shooting medium
Examples of transgenic crops.
Reduced environment pollution
→ crop sprayed only 1 with broad specific herbicide
→ herbicide tolerant Sb the ↓ in herbicide 10 - 40%
Habitat preservation
→ need only single herbicide = spraying con be - delayed
→allows weeds to grow and provide habitant food for lots of invertebrae important → -soilecosystems
Eg of transgenic crops ) →
Cotten , 1 rice tomato
Potential genetic transfer problems?
Could herbicide tolerance transfer to wild relatives or conventional crops
Crops need a lot of “care “ and have poorsurvivability in the will d
Hybrids between crops s weeds
Crops can be isolated via buffer zone
2 types of gene therapy?
Germ line therapy = inserting DNA into a fertilised egg _ are cells including gonads contain the gene
Illegal IN humans
Somatic therapy = inserting DNA into somatic cells affected by disease
