Module 03 - Section 04 Flashcards
Amplification of DNA by polymerase chain reaction
What is molecular cloning?
Isolation and generation of recombinant DNA molecules that are placed in organisms for replication or study.
What is Recombinant DNA?
DNA formed by joining DNA molecules, sometimes from different species in different combinations
What does a simple PCR mixture contain? (4)
(1) DNA sample containing the segment to be amplified
(2) Pair of synthetic oligonucleotides primers
(3) dNTPs
(4) DNA polymerase
What are the 5 steps of the PCR procedure?
(1) Denaturation
(2) Annealing
(3) Elongation
(4) Amplification
(5) Repeat
Describe the Denaturation step in PCR procedure
To denature of the DNA duplex, the reaction mixture is briefly heated, separating the strands. This enables the components of the reaction to interact with the single-stranded DNA template
Describe the Annealing step in PCR procedure
The mixture is cooled to that the synthetic primers can anneal to the DNA template. The high concentration of primers increases the likelihood that they will anneal to each strand of the denatured DNA before the 2 strands can re-anneal to one another
Describe the Elongation step in PCR procedure
Temperature is slightly increased again to allow for synthesis of a complementary DNA strand. DNA polymerase recognizes the 3’ ends (free 3’-OH) of the hybridized primers and carries out DNA synthesis to extend these strands along the targeted segment
Describe the Amplification step in PCR procedure
This process is then repeated for a second time , reaction mixture is heater to denature the DNA and cooled to allow the primers to anneal. Strands are elongated to amplify the replicon
Describe the Repeat step in PCR procedure
The cycle of heating cooling and extension is repeated 25-30 times over a few hours in an automated process. Each cycle doubles the amount of the amplified DNA segment, so it grows exponentially.
Equation: 2^n, where n is the number of cycles
What are some applications of PCR? (2)
(1) Paleontology: cloning of rare partially- or un-degraded DNA segments from samples - ex: woolly mammoth
(2) Epidemiology: Trace evolution of human pathogenic viruses through PCR enhanced DNA samples from humans
What is a serious issue due to the extreme sensitivity of PCR methods?
Contamination of samples - controls must be run to make sure the amplified DNA is not derived from the researcher or from contaminating organisms
What are the parameters of designing a food PCR primer set? (6)
(1) 18-25 nucleotides in lenght
(2) 40-60% GC content
(3) Annealing temperature in the range of 50-60 degrees
(4) 1 or 2 residues at the 3’ end of the DNA strands
(5) Minimal secondary structure
(6) complementary to sequence chose for amplification
What is the equation for calculating melting temperature for dsDNA fragments?
Tm = 0.41 (% G+C) + 69.3
What is equation to calculate shorter oligonucleotides? (aka primer)
Tm = 2 (A+T) + 4 (G+C)
What is the equation for the annealing temperature?
Ta = Tm - 5
What is the goal of gel electrophoresis?
separate mixtures, by size, of large charged molecules such as proteins and nucleic acids
What is the mechanism of Gel Electrophoresis?
(1) Sample of interest is added to a slot at ont end of the gel (agarose = does not disrupt nucleic acid base pairing)
(2) Voltage is applied to the gel. Because the backbone of nucleic acids is negatively charged, both RNA and DNA will migrate towards the positive end of the gel in the electric field
(3) Larger molecules tend to more more slowly than smaller ones. Larger molecules stay closer to the top while smaller molecules migrate further towards the bottom
How is the PCR product visualized ?
Gel is stained with a molecule called ethidium bromide
What is ethidium bromide?
Planar molecule that is abled to act as an intercalating agent (inserts itself in between nucleic acid strands in a nonspecific manner) and fluoresces when exposed to UV light
Describe briefly the mechanism of Reverse Transcriptase
Amplifies RNA, by converting RNA to cDNA which is then amplified by PCR
Why use RT-PCR? (2)
Can be used to amplify and sequence gene segments without introns
Can be used to quantify mRNA levels as a measure of gene expression using a process known as quantitative PCR (qPCR)
What is the difference between qPCR and end-point PCR?
End-point PCR: reactions are assessed after 35-40 rounds of amplification, but aren’t quantified
qPCR: product generated by the reaction is quantified after every reaction cycle
Describe the process of qPCR (4 steps)
(1) DNA is denatured at 95 degrees in a solution containing SYBR green
(2) Primer annealing (55-65 degrees)
(3) Extension (60-72 degrees)
(4) Special thermocycler records fluorescent emission from the reaction (SYBR green)
What is the exponential phase of qPCR?
Phase where exponentially more fuorescence can be detected with each cycle
What is the plateau phase of qPCR?
Signal/ fluorescence reaches a “max” or a plateau, because one or more reaction components are exhausted
What is the Cycle Threshold (Ct)?
Cycle number at which the threshold is first surpassed. The threshold is the cycle at which the qPCR reaction will enter the exponential phase
Which will reach the threshold faster; a sample with high concentration or low concentration?
high concentration
