PL3 Flashcards
what are the fundamental techniques in molecular biology
polymerase chain reaction (PCR)
DNA sequencing
describe PCR (polymerase chain reaction)
amplifies a specific DNA sequence which can be part of a complex mixture
does not have to be purified
name 4 uses of PCR
sequencing (some approaches) - study sequence of that section
DNA cloning (isolating a particular gene) - to study it in more depth
detection of pathogens (like SARS-CoV-2)
gene editing (edit an amplified gene)
what does PCR generally depend on
knowing the nucleotide sequences at the ends of the region to be amplified
what does single reaction tube contain
DNA template (can be complex mixture, like total DNA from cell)
DNA polymerase that is stable at high temp (to make copies)
primers complementary to each end of the region to be amplified (oligionucleotides or oligos)
dNTPS (monomers to create new polymers)
does DNA need to be denatured for PCR and explain why or why not
yesss
heat destabilizes the double helix
need to be separated to use as templates
hot temp used - depends on G-C content - more G-C = higher temp since 3 H bonds (harder to break)
what is 3 step cycle of PCR
denaturation
annealing of primers
extension by DNA polymerase
how many repetitions are needed for PCR
20-40
describe denaturation (step of PCR)
temp around 95 or 98 degrees
hot enough regardless of G-C content
DNA strand separate
describe annealing of primers (step of PCR)
temp decreased to allow primers to base pair to complementary DNA template
anneals to each end of segment to be amplified
~50 degrees
describe extension by DNA polymerase (step of PCR)
polymerase extends primer to form nascent DNA strand
around 70 degrees
synthesis of new strand
describe thermal cycler
can rapidly change temp
used for PCR
hot plate with test tubes - fast at changing temp
before PCR was harder
describe the DNA primers (how they are made)
oligonucleotides are designed by computer to be complementary and specific to ends of the sequence to be amplified and commercially synthesized
cost very low <1dollar
which DNA polymerase is used for PCR
Taq polymerase
where does Taq polymerase come from
thermophilic bacterium (thermus aquaticus)
why is Taq polymerase good for PCR
thermally resistant so enzyme will not denature at high temp
cheap but no proofreading activity so it is best for amplifying short fragments
other enzymes with higher fidelity (accuracy) can also be used
what does exponential amplification allow for
a very sensitive detection of DNA sequence in the sample and enables purification of substantial amounts of a specific fragment for further use
why does PCR amplify DNA
each time you have more template strands
can you keep doing PCR forever
primers and dNTPs can run out so no
can primers anneal the wrong way
yessss s
can be created wrong
creates more off target effects
what method can analyze DNA fragments produced by PCR
gel electrophoresis
describe gel electrophoresis for PCR
solid like jelly = gel
they move inversely to length
separation based on size
if you wanted to amplify 1.5kb then you run electrophoresis and there should be a prominent band of DNA at 1.5kb - way to tell if it worked
what is a method of DNA sequencing
dideoxy chain termination method of DNA sequencing
classical sanger sequencing
is classic sanger sequencing still used
primary method from late 1970s to 2010 around
sometimes still used for verification of individual results
