Genetic Tools Flashcards
What is PCR?
a test for the amplification of DNA
what are the 3 steps in PCR?
- Denaturation
double DNA strands melts open - Annealing
primers bind to DNA, polymerase starts copying DNA - Extension
at 72oC
repetition of pcr
heat and then cool
allows primers to bind to DNA
cycles repeat
30-40 cycles
Amplification
Only DNA between primers is amplified
ends are identified by the primers added
What are the PCR primers?
short
20bp
single stranded DNA
chemically synthesised
what is agarose gel electrophoresis?
lab technique
provides matrix for DNA
speed varies depends on size and shape of the product
lighter move faster than heavier
separation of DNA by size
Potential difference along the gel
moves to positive electrode
dependent on conformation & size
stain DNA with fluorescent dye for detection by UV exposure
What is Sanger Sequencing?
Mix of different length DNA strands
each terminating with fluorescent nucleotide
run through capillary tube & at end there is a laser reading
smallest first then larger will read
What are the uses of PCR?
detection of pathogens in water
dna sequencing
genetic fingerprinting
forensic analysis
diagnosis of genetic disorders
What are the limitations of PCR?
sequence information required
limit on length
potentially high error rate
very sensitive to exact reaction conditions
need primers so need background info
Key points of PCR
use for in vitro DNA synthesis
requires prior knowledge
gel electrophoresis used to visualise
broadly used lab technique
what are plasmids?
plasmids are small extra chromsomal dna
& can be shared between bacteria
How do you cut DNA?
by restriction endonucelases
creating 5’ overhang
How do you join DNA?
DNA ligase
forming phosphodiester bonds
requiring ATP
What is recombinant DNA?
a set of dna that is within a cloning vector
1. cloning vector cleaved by restriction nuclease
2. covalent linkage of DNA fragment by DNA ligase
3. creating of recombinant DNA