where do we extract DNA from?
- blood
- body hair
- bones
-teeth - mucus
- saliva
- tissue cells
- semen
how is DNA extracted and amplified?
via a process called Polymerase Chain Reaction
why do we extract DNA?
- forensic science
- paternity testing
- family ancestry
- disease testing/ screening
- wildlife conservative
- archaeological findings
How does DNA extraction take place?
- cells in the sample are broken open - by blending or grinding
- fats and proteins in the mixture are emulsified -adding detergent and salt
- ethanol is added and the sample is centrifuged to separate DNA from the rest of the cell debris
- extracted DNA can be amplified, sequenced and analysed
What is the purpose of PCR?
used to amplify extracted DNA fragments
what are the applications of PCR?
- gene isolation
- sequencing
- cloning
- mapping mutations
- studying gene expression
- diagnostic testing
How does PCR work?
- temperature changes within the machine start DNA replication using primers
- fragments are doubled each cycle
What tools are used in PCR?
- PCR machine
- DNA polymerase
- primers
- nucleotides
- sometimes fluorescent probes are used
what do we need for PCR?
- water and buffer
- magnesium
- nucleotides
- primers
- DNA polymerase
- DNA Template
What is taq polymerase?
- DNA polymerase builds new strands of DNA
- used with PCR
- originally isolated from Thermus aquaticus bacteria
very heat stable as naturally found in thermal hot springs - most active around 70°C
What are the 3 PCR changes?
- denaturation
- annealing
- extension
when does denaturation occur?
at 90°c
when does annealing occur?
- primers bind to single-stranded DNA
- 55°c- 65°c
when does extension occur?
- polymerase extends primers
- 72°C
what is DNA profiling?
- where a specific DNA pattern called profile is obtained by bodily tissue
- determines individual DNA characteristics
how does DNA profiling work?
-uses restriction enzymes cut individual DNA into fragments
- fragments separated by electrophoresis
what is gel Electrophoresis?
- used to analyse genes and proteins
- separates molecules
- movement stimulated by electric current
- different segments travel different distances depending on size
what is movement of the charged molecule called?
migration
What is agarose gel used for?
- holds DNA
How is agarose gel prepared?
by combing agarose powder with buffer soloution
what is agarose gel?
- insoluble at room temperature
- dissolves when heated
boiled until clear and poured into tray to set
what happens during gel electrophoresis?
- edges of tray sealed and comb inserted
- agarose gel cools it will polymerise and form a flexible gel
- once gel has cooled completely the combs can be removed leaving behind a set of ‘wells’ in the gel
