PCR Flashcards
PCR origins
PCR Origin
allsed to determine har changes in genome sequence lead to dounstream changes in gene expression and physiology
• Kary Mullis - was awarded a Nobel Prize in Chemistry in the year 1993 for inventing the PCR technique
• PCR is a method of DNA cloning
• It is a rapid method that does not require host cells for DNA replication
• Can amplify target DNA sequence from tiny amounts of DNA
-Used to determine how changes in sequence lead to downstream changes in gene expression and phenotype
PCR Components
- Template DNA
- Primers - short oligos 20 - 25 nt in length
- Artificially designed
- some aspects of the sequence to be amplified must be known
- dNTPs - deoxynucleoside triphosphates
- free nucleosides that can be incorporated into the replicated DNA fragments by use of the polymerase
-DNA polymerase - an enzyme that is involved in replicating the target DNA fragments
* It must be heat stable so that it can remain functional during thermocycling
-DNA polymerases - isolated and cloned from thermophilic bacteria such as Thermophilus aquaticus (Taq) and Pyrococcus furiosis (Pfu) are used in PCR
-Other components - Mg2* or ions are co-factors or buffer pH regulator
Cofactor
It helps the active site of an enzyme be exposed to the substrate or allows it to conform. It is mostly inorganic
Oligo
Another word for primer and means the nucleotide is artificially synthesized
Amplicord
It is the final product in test tube after PCR occurred
PCR process
PCR requires two primers
• Primers: Short, single-stranded oligonucleotides, one complementary to 5’ end and another complementary to 3’ end
• dsDNA to be cloned is put in tube with DNA polymerase, Mg?+, and dNTPs
• Primers anneal to denatured DNA
• Complementary strands are synthesized by heat-stable DNA polymerase
• e.g. Taq DNA Polymerase isolated from thermophilic bacteria
PCR steps
• Three steps of PCR
• Denaturation - heat to 94 - 95°C to allow DNA template strands to separate
• Primer annealing (hybridization/annealing) - reduce temperature to Tm - 5°C to allow for primer binding
• Extension - Temperature suitable to the polymerase (72°C) to allow the strand to be extended
• Steps are repeated over and over using thermocycler to amplity DNA exponentially
• DNA strand is doubled in each cycle
• New strands along with old strand serve as templates in next cycle
Target DNA Amplification
• Target DNA sequence doubles in each cycle
• Number of copies of the
fragment = 2”
• Where n = number of cycles
• By 25 cycles, you have 225 = approx. 33,5 million copies in the reaction tube
Setting up a thermocylcer
• Thermocycler is set up to have an initial Denaturation step that allows for double stranded DNA to separate.
• It then cycles through denaturation, annealing and extension for between
25 - 35 cycles.
• There is a final elongation step to ensure that all strands are replicated prior to termination.
• The reaction is terminated by reducing the temperature to 4°C.
• PCR products are stored by freezing samples at -20°C.
Setting up a thermocylcer
• Thermocycler is set up to have an initial Denaturation step that allows for double stranded DNA to separate.
• It then cycles through denaturation, annealing and extension for between
25 - 35 cycles.
• There is a final elongation step to ensure that all strands are replicated prior to termination.
• The reaction is terminated by reducing the temperature to 4°C.
• PCR products are stored by freezing samples at -20°C.
Capillary electrophoresis
• Capillary electrophoresis is a gel free method for nucleic acid separation
• Uses the property of the capillary tube - diameter of approximately 50 um
• Fragments move through the capillary tube at different speeds due to their electrophoretic mobility
• Positively charged particles move fastest and negatively charge particles move slower as their movement is impeded by the anode
Duke to negatively charged phosphate backbone, larger DNA fragments move slower than smaller ones
• A detector placed along the capillary tube can detect UV or fluorescent signatures in the tube
• These measurements are recorded over time and represented as an electropherogram
It is faster than agarose gel, less labor intensive and does not need buffers
Negative PCR control
It contains all the same substances that a normal PCR has except for DNA.
It checks for contamination.
PCR limitations
• Some information about nucleotide sequence of target DNA is required to synthesize primer
• Minor contamination from other sources can cause problems (e.g., skin cells from researcher)
• PCR cannot amplify long segments of DNA (maximum 10 kb)
- problem with primers: If a target is rich in CG, primers may anneal too strongly and if it is rich in AT then it anneals too weakly
PCR Applications
PCR is the most widely used technique in genetics and molecular biology
• Allows for screening of mutations involved in genetic disorders
• Location and nature of mutation can be determined quickly
• Allele-specific probes for genetic testing can be synthesized, making PCR important for diagnosing genetic disorders
• It is used in genotyping and genetic variation studies
• It is also used for gene expression studies
Variations to PCR
Variations to Standard PCR
• Reverse Transcriptase PCR (RT-PCR)
• PCR technique modified to amplify RNA rather than DNA
• Involves reverse transcription of RNA to cDNA
• Real Time/quantitative PCR (qPCR)
• A quantitative method that relies on fluorescent probes to determine the amount of DNA/copy number that is present in a sample.
• This is often combined with RT-PCR to quantify mRNA expression
• Multiplexing
• Multiple PCR products are amplified in one reaction tube
• They are individually labelled with probes bound to primers and detected on a gel or via capillary electrophoresis
