Manipulating Genomes Flashcards
What is the polymerase Chain Reaction used for
To generate many copies of DNA fragments and amplify it
How is PCR done
Double stranded DNA is heated to 95C to break hydrogen bonds between bases
Primers are added and temp is lowered to 55C to allow primers to anneal
Temp raised to 75C and Taq DNA polymerase binds and extends primers using free nucleotides
Repeat for exponential growth
What are common techniques for studying genes
Polymerase chain reaction
Cutting out DNA fragments
Gel electrophoresis
What is PCR used for
To amplify DNA from crime scenes, archaeological samples, during DNA sequencing
What are restriction enzymes used for
Used to obtain DNA fragments from a genome
How do restriction enzymes work
Some sections of DNA have palindromic sequences, restriction enzymes recognise specific sequences and cut at these sites - leaving sticky ends
Different restriction enzymes cut at different sequences as shape of sequence is complimentary to shape of active site
What is Gel electrophoresis
Use of electrical current to separate DNA fragments by size
How does gel electrophoresis work
DNA fragment laid in electrophoresis tank and covered with buffer solution
Nucleotides are negatively charged and move towards anode
Larger fragments move slower than smaller ones
What gel is used for gel electrophoresis
Agarose
Why is agarose gel used in gel electrophoresis
Has large pores
What needs to happen to proteins to enable them to be used in gel electrophoresis
Needs to be natured in order for them all to have the same charge
What is the chain termination metjod
Enables us to determine nucleotide sequences on DNA fragments, sequencing mixture then goes through PCR
What is in the sequencing mixture
Original DNA, Primers, DNA polymerase, DNA free nucleotides - some have fluorescent markers
Why do some of the DNA free nucleotides used in the chain termination method have a fluorescent marker
The DNA has been modified to prevent replication and are fluorescent
4 different colours correspond to the 4 different bases
Allows us to determine sequence of bases
What is the downside of the chain termination method
Can only be used on DNA fragments smaller than 1000 base pairs
How are genomes sequenced
Restriction enzymes (RE) cut DNA into smaller fragments
RE cut plasmid out of BACs so it can be used as a gene libraries for the DNA fragment
BAC segments are then removed and cut into smaller fragments by RE
PCR amplifies the DNA
Each fragment is sequenced and then the sequences of overlapping segments are compared by computers
Sequence of whole BAC segment is determined
What are BACs
Bacterial artificial segment
What are the uses of sequencing
Allowed for the sequence of amino acids in a polypeptide chain to be predicted
Development of synthetic biology
Developed computational biology and bioinformatic
Helps determine evolutionary relationships
Useful for epidemiology
What is genetic engineering
A process which involved modifying the genome of an organism by introducing a gene from another organism
What is a transgenic organism
contains DNA from another organism
What is the process of genetic engineering
1) DNA containing the desired gene is obtained using a restriction enzyme (RE)
2) Recombinant DNA is made
- Vector DNA is isolated, cut open using same RE so that the sticky ends are complimentary
- DNA fragment inserted into vector DNA using DNA ligase
3) vector is used to transfer recombinant DNA into bacterial cells
How do bacteriophage with recombinant DNA transform cells
Bacteriophage injects DNA into bacterial cell which integrate with bacterial DNA
How do plasmid vectors with recombinant DNA transform cells
Plasmid are placed into a mixture with host cells
Electric field is created - increasing permeability of host plasma membranes
Plasmids enter by electroporation
Features of GM soybean
GM soybean contains a gene that codes for a protein that is a toxic to some insects
Gene taken from bacillus thuringienisis and put into plasmid from Agrobacterium tumefaciens
