molecular techniques Flashcards
what is PCR?
polymerase chain reaction
PCR is used to amplify a specific region of DNA from a trace sample
what are the 5 components (ingredients) of PCR?
- DNA template - contains nucleotide sequence of interest/gene of interest
- PCR primers - 2 sets of short, single-stranded DNA primers that are specific to the sequence of interest. the primers flank the sequence of interest and are complementary to the 3’ end of both template strands.
- free dNTPs - must be present in excess as raw materials for the synthesis of new DNA strands
- thermostable DNA polymerase - eg Taq polymerase must be stable at high temperatures and is not denatured by repeated heat treatments
- PCR reaction buffer - eg Mg2+ ions function as the cofactors required for DNA polymerase activity
DAE
what are the 3 steps in a single cycle of PCR and the respective temperatures they should be carried out at?
- denaturation of DNA template -> 90C-100C
- annealing of primers -> 50C-65C
- extension of primers -> 60C-75C
^^ in chronological order!!
describe the denaturation of DNA template during PCR.
the reaction is heated to 95C for 30s.
at this temp, hydrogen bonds holding the 2 strands of the DNA template are broken -> DNA template is denatured and becomes single stranded DNA
describe annealing of primers during PCR
the reaction mixture is cooled to 54C for 1 min in the presence of a large excess of the 2 sets of DNA primers
cooling allows primers to anneal specifically to complementary sequences at the 3’ end of single-stranded DNA templates via hydrogen bonds (primers provide 3’ OH group for DNA polymerase to elongate)
annealing of primers to their complementary DNA sequences is also referred to as hybridisation
the optimum annealing temperature for a pair of primers depends on primer length and base composition
describe the extension of primers in PCR
the reaction mixture is heated to 72C for 2 min, which is close to the optimum temperature of the thermostable Taq polymerase
the annealed primers prime DNA synthesis using the 4 dNTPs. the process is catalysed by Taq polymerase
the region of DNA downstream of each primer is extended in the 5’ to 3’ direction
why is PCR a chain reaction?
PCR is a chain reaction because newly synthesised DNA strands will serve as templates for DNA synthesis in subsequent cycles.
within a few cycles, the predominant DNA species is identical to the sequence of interest, the sequence flanked by and including the 2 primers
PCR is highly specific as only the sequence of interest is amplified because primers are specific and do not attach elsewhere
number of DNA molecules after n cycles can be calculated by taking 2^n
SSR
what are the advantages of PCR?
- sensitivity - PCR is extremely sensitive, and can amplify sequences from minute amount of DNA
- speed & ease of use - PCR is rapid and can be easily automated. a single cycle takes less than 5 minutes
- robustness - PCR can permit amplification of specific sequences from material in which the DNA is badly degraded
RISNO
what are the limitations of PCR?
- risk of contamination - due to extreme sensitivity of PCR, any contamination by non-template nucleic acids could cause non-target sequences to be amplified instead
- infidelity of DNA replication in vitro - DNA polymerases used in PCR often lack 3’ to 5’ exonuclease activity (for proofreading DNA) so errors may be wrongly incorporated
- short size and limiting amounts of PCR products - DNA polymerases efficiently amplify DNA products up to a few thousand base pairs. PCRs for longer products are less efficient due to enzyme activity loss and inaccuracies accumulated (as DNA polymerases cannot proofread)
- need for target DNA sequence info - prior sequence information is needed to construct specific oligonucleotide primers that permit selective amplification of a particular DNA sequence
- only can be applied to amplify nucleic acids - PCR cannot be used to amplify proteins
what is gel electrophoresis?
gel electrophoresis separates charged molecules, eg nucleic acids and proteins, based on their different rates of movement/migration (due to differing size) in an electric field
what can gel electrophoresis be used for?
- to separate DNA fragments according to size
- to determine the approximate molecular weight of the separated DNA fragments (need to use DNA ladder)
- to isolate/purify individual DNA fragments for further study
- to check the results of PCR (if PCR is successful, only 1 band should be present after gel electrophoresis)
PSLAS
what are the 5 steps of gel electrophoresis?
- preparation of gel with walls for DNA samples
- setting up of gel for electrophoresis
- loading of DNA samples into wells
- application of electric field to start electrophoresis
- staining of gel to view separated DNA bands
describe the process of gel electrophoresis
usually not tested so just read through
- preparation of gel with wells for DNA samples - agarose powder is mixed with a buffer solution, which helps maintain the DNA in a stable form during electrophoresis. the mixture is heated until the agarose dissolves. a gel tray and gel comb is placed at one end to create wells in the gel, which DNA will subsequently be loaded into. the gel solution is poured into the gel tray and allowed to cool and solidify
- setting up of gel for electrophoresis - after the gel has solidifed, the gel tray with the solid gel is placed in an electrophoresis chamber filled with enough buffer to cover the gel, allowing the current to flow through the gel. the gel comb is removed, leaving wells for the loading of DNA samples
- loading of DNA samples - DNA samples are mixed with a loading dye that makes the DNA sample visible for easier loading into wells. each DNA sample is loaded into an individual well, with one well being reserved for the molecular weight marker (DNA ladder) which allows for size determination of the bands
- application of electric field - a DC power supply is connected to the electrode and turned on. DNA is forced to move by the application of a DC from the negatively charged cathode towards the positively charged anode. electrophoresis is conducted until DNA molecules of different sizes are well separated
- staining of gel to view separated DNA bands - DNA molecules separated in the gel are only visible after staining the gel with DNA-binding dye like methylene blue or ethidum bromide. viewing of bands can be accomplished by autoradiography if the DNA is radioactively labelled
in step3, loading dye consists of bromophenol blue, xylene cyanol and glycerol. bromophenol blue and xylene cyanol move at different rates, which allows the monitoring of the rate of DNA movement. glycerol is added to increase the density of the DNA sample so they sink to the bottom of the wells
how does the gel help in gel electrophoresis?
- the semisolid, porous gel matrix separates DNA molecules based on their rate of movement through the gel matrix (since phosphate groups in DNA are negatively-charged, DNA moves towards the positive electrode
- the complex network of pores in the gel matrix act as a ‘molecular sieve’ to impede the movement of DNA molecules and separate them by size/length
- shorter DNA fragments are less impeded by the pores and move through them more quickly. DNA fragments of diff lengths migrate as distinct bands, so a complete mixture of linear DNA fragments is size-fractioned into discrete bands
honestly idk if this is important but js read through
after electrophoresis, the gel is removed and stained with a DNA bindin dye (eg methylene blue or ethidium bromide) to visualise the separated DNA fragments as a series of bands within a gel
what is nucleic acid hybridisation?
nucleic acid hybridisation is the process by which 2 complementary, single-stranded nucleic acid chains base-pair and reform a double-stranded hybrid
can occur between any 2 single-stranded nucleic acid chains, as long as they have complementary nucleotide sequences.
used to detect specific DNA and RNA base/nucleotide sequences using specific single-stranded nucleic acid probes of known sequence
