PCR Flashcards
Polymerase chain reaction
Used to make many copies of a specific DNA region in vitro
DNA polymerase
Reads template DNA and adds complementary nucleotides in 5 to 3 direction
Taq pol
Has a high optimum temp and so can stay stable at high temps
PCR components
DNA pol, DNA template, primers, free nucleotides, free ions and sterile water
DNA template
Can be genomic or complementary DNA
Genomic DNA
Very stable and it identifies mutations and genetic markers
Complementary DNA
Made from RNA, it shows if a gene is being transcribed and the response of genes to treatments
Primers
Short ssdna fragments that direct pol to where it needs to bind which ensures that only the region of interest is being copied
Primer design
Length of 18-30 bps, gc content of 40 - 60 %, and annealing temp is 50-65°C
What to avoid in primer design
4 + runs of the same base, repeat pattern of 2 bases, self-complementary primer and homology of forward and reverse primers
Annealing temp
Temp at which primer duplexes with ss DNA
Mispriming
When the primer anneals to an unintended location
Free nucleotides
Deoxyribonucleotide triphosphates that are recruited by DNA pol to synthesize the new strand
Free ions
Maintain enzyme-substrate complex and buffer the ph
K+
Provides stability for primer binding
Mg 2+
Catalyse phosphodiester bond and stabilizes complex of pol + primer + template
PCR program
Initial denaturation, denaturation, annealing, extension, final extension and cooling for 25 - 35 cycles
Denaturation
Separation of supercoiled DNA by breaking h-bonds at 95°C
Annealing
Primers bind to template at 50 - 65 °C
Extension
Taq pol extends primers and the new strand is synthesized at 68 - 72°C
Thermocyclers
Carries out PCR because it can be programmed to run at different temps
Visualizing PCR
Determine DNA fragment size by gel electrophoresis then visualize the results using a dye and UV light
Optimizing PCR
By adjusting primer annealing temp, annealing time, extension time, mg 2+ conc, k+ conc, amount of template or amount of pol
High annealing temp
More specific primer binding
Mg 2+ conc
Adjusting it affects the number of bands produced
Conventional PCR
Non-quantifiable amplification of g DNA that results in a single product
Reverse transcriptase PCR
Convert RNA into cDNA, it is semi-quantitative and used to determine if the mRNA transcript is present
Quantitative real time PCR
Amplification and quantification of cDNA and gene expression by quantifying the fluorescent signal produced during PCR
Low cycle number indicates a high transcript amount
Microarrays
Detects thousands of genes simultaneously and investigates mutations in genes and determines if they’re on or off
