Biotechnology Flashcards
DNA tools
- restriction enzymes
- DNA ligase
- DNA polymerase
- Primers
Restriction enzymes
- moves along DNA until finds recognition site - cuts at the recognition site
creates smaller pieces, called restriction fragments
two types of cuts
blunt end cut
straight cut, no overhang
sticky end cut
cuts at different spots
more often used
one strand has overhanging complementary bases
DNA ligase
important in DNA replication
seals/reassembles DNA fragments - ligation
DNA polymerase
class of enzymes that synthesise new strands of DNA
adds free nucleotides to make a new strand
used in amplifying DNA during PCR
Primers
short fragments of single stranded DNA or RNA
signal for the polymerase to begin synthesis
DNA base techniques
methods to analyse, modify and manipulate DNA bases
Amplification
- increase number of copies of a DNA sequence for further lab use
- most common method is PCR
PCR - polymerase chain reaction brief description
amplifies a specific DNA sequence by repeatedly heating and cooling the sample to enable DNA denaturation, primer binding, and enzymatic replication, producing millions of copies
- replicated many times
- increase copies a lot
- has requirements
PCR - 1. Denaturation
- DNA double strand is heated up to around 95°C to separate into single strands by breaking the bonds between the bases
PCR - 2. Annealing
Temperature is lowered to 50 - 60°C so primers can bind to their complementary sequences, by joining back the H bonds
PCR - 3. Extension/elongation
DNA taq polymerase attaches free nucleotides to the end of primers - can not work at high temperatures - 72°C
PCR - 4. Repeat cycle
These steps repeated for multiple cycles, exponentially amplifying the target DNA sequence
e.g. 4, 8, 16, 32
Gel electrophoresis
- extract DNA samples to be analysed from an organism’s cells, than cut the strands withe restriction enzymes
- pour agarose, jelly like material into a tray, solution sets as gel
- make wells in gel and add a buffer solution, this solution covers the entire gel and the wells imbedded
- place DNA samples in well with the use of pipettes
- run a current through the gel electrophoresis machine, through the gel, molecules (because negative) travel towards the positive end, the smaller molecules move faster (negative molecules are repelled by positive electrode and travel away from beginning)
- analyse the data and any patterns shown by the gel electrophoresis - under ultraviolet
