8.4.1 Recombinat DNA Tech Flashcards
(17 cards)
What is recombinant DNA tech
Transfer of DNA fragments from one organism or species to another
How can DNA fragments be produced
Restriction enzymes cut DNA at specific recognition sequences, at either side of desired gene:
-shape of receptor recognition is complementary to active site
Staggered = sticky ends
How can DNA fragments be produced from mRNA
Isolate mRNA
Mix it with DNA nucleotides and reverse transcriptase (creates cDNA)
DNA polymerase can form the 2nd strand of DNA using cDNA as a template
Advantages of using mRNA to produce fragments
Much more mRNA in cells than DNA
No introns in mRNA
How can fragments be produced using a gene machine
Snythesisi fragments quickly and accurately from scratch
Amino acid sequence is proteins is determined
Don’t contain introns
In vitro and in vivo techniques to amplify DNA fragments
In vitro (outside living organism):
-polymerase chain reaction (PCR)
In vivo (inside living organism)
Using bacteria
Explain PCR
Mixture heated to 95:
-seperated DNA strands
-breaking H bonds between bases
Cooled to 55:
-primers can now bind
- forming H bonds between bases Cooled
Heated to 72:
-nucleotides align next to complementary exposed bases
-DNA polymerase joins adjacent DNA nucleotides forming phosphodiester bonds
Role of primers in PCR
-short single stranded DNA stands
-complementary to DNA
-allowing DNA polymerase to bind
-2 different primers are needed
Why would DNA replication eventually stop with PCR
Limited number of primers and nucleotides
Summarise amplifying DNA fragments in vivo (bacteria)
Add promoter and terminator regions to DNA fragments
Inserted DNA fragments and marker genes into vectors (plasmids) using restriction endonuclease and DNA ligase
Transform host cells by inserting the vectors
Detect the genetically modified organism by using the marker gene
Culture the bacteria (they divide into clones)
Use of promoter regions and terminator regions
Promoter:
-allows transcription to start by allowing DNA polymerase to bind to DNA
-ensures gene expression only happens to specific cell types
Terminator:
-ensure transcription stops
Role of enzymes in vivo
Restriction endonuclease cuts vector DNA(sticky ends for complementary base pairing)
DNA ligase joins DNA fragments to vector DNA (forming the phosphodiester bonds)
Why are maker genes used
To Detect the GM organisms
As not all cells will take up the vector
How can recombinant DNA be useful
Medicine:
-produce insulin
-gene therapy
Agriculture:
-GM crops
Industry:
-produce enzymes used in industrial processes
What is gene therapy
Introduction of new DNA into cells (containing healthy alleles)
To overcome faulty cells
Issues with gene therapy
Effect is short lived- requires regular treatment
-long term affect not known
Negative effects of recombinant DNA
Could affect food webs reducing biodiversity
