DNA Technology Flashcards
Recombinant DNA
laboratory manipulation of DNA in which DNA fragments from various sources are cut, combined, and reinserted into living organisms
Animal Examples of Recombinant DNA
Mice - used to study human immune system
Chickens - more resistant to infections
Cows - high milk supply and leaner meat
Plant Examples of Recombinant DNA
disease/insect resistant crops
Bacteria Examples of Recombinant DNA
make human insulin/growth hormone
Restriction Enzymes
used to cut DNA into fragments
restriction enzymes identify and cut specific sequences of base pairs on DNA molecules (recognition site)
- look for palindrome sequences
blunt end cut
restriction enzymes make a clean cut and the fragments do not have an overhang
sticky end cut
restriction enzymes make a staggered cut and the fragments have an overhang
How are sticky ends used to join two fragments of DNA together?
two DNA fragments can only be joined together if they have been produced using the same restriction enzyme
when two fragments of DNA initially combine, they are held together by hydrogen bonds
DNA ligase is used to complete the fusion by forming phosphodiester bonds between the strands
Plasmids
Small circular pieces of DNA found in bacteria
Plasmid is cut with the same restriction enzymes as the target gene segment and placed in solution
Matching sticky ends connect
Bacteria will now express the inserted gene
What is Gel Electrophoresis?
process used to separate DNA fragments based on their relative lengths
Process of Gel Electrophoresis
- DNA is treated with restriction enzymes to cut them into various lengths, then placed in wells
- Because of DNA’s negative charge, when the current is on, DNA will move towards the positive electrode
- Small fragments will move through the gel faster than large fragments, causing separation of different fragment sizes
What is Polymerase Chain Reaction (PCR)?
used to make multiple copies of a DNA fragment
Process of PCR
- Begins with a solution of DNA fragments, DNA primers, Taq polymerase, and free nucleotides
- Heat used to separate the two strands of DNA (breaks H bonds)
- Then, the mixture is cooled and the DNA primers will anneal to the strands of DNA
- Taq polymerase starts at the primers and builds the complementary strands of DNA using the free nucleotides
- Repeat
Sequencing DNA
Importance
complete sequence for gene helps understand gene function
- identify mutations
- locate regulatory sequences
Process of Sequencing DNA
- DNA strand is copied through PCR
- 4 test tubes are prepared with: DNA polymerase, primer and strands, normal nucleotides, labelled dideoxynucleotides
- DNA replication begins - Nucleotides are added to building strand until dideoxynucleotide is attached
- prevents any other nucleotides from being added on to the strand (marked with fluorescence) - Fragments of varying lengths are generated which are then separated using gel electrophoresis
