Aging Aging

Mitochondrial DNA Mutations 2593. PCR amplification: For each concentration of serially diluted DNA (1 µL), set upthe following reaction:35.8 µL Sterile water5 µL 10× GeneAmp ® reaction buffer5 µL 2 mM dNTPs1.5 µL L3108 or L82821.5 µL H3717 or H138321 µL Template DNA0.2 µL AmpliTaq® DNA polymerase (see Note 15)When setting up a number of PCR reactions to investigate a number of concentrations,make an appropriate volume of a PCR master mix for each primerpair containing all the components except template DNA. Aliquot these into therespective tube, and add 1 µL of the appropriate serially diluted DNA.4. Perform 34 cycles of amplification as follows:Initial denaturation 94°C 2 min34 Cycles 94°C 45 s51°C for 30 s (wtDNA) or56°C for 30 s (mtDNA 4977 )72°C 1 minFinal extension 72°C 8 min5. Electrophorese the PCR products (40 µL) through a 1.5% agarose gel in 1× TAEbuffer using a large horizontal electrophoresis unit.6. Visualize bands by UV transillumination.7. Quantitation of PCR: The gel image is stored using a digital imaging system, andthe optical densities of the PCR products are quantified using image analysissoftware. An integrated density value (IDV) for a set area is obtained for eachPCR product. The DNA concentration at which the IDV is zero is obtained forthe wtDNA and mtDNA 4977 , respectively. The percentage of mtDNA 4977 deletionin the DNA sample is calculated by dividing the DNA concentration at which thewtDNA IDV is zero by the DNA concentration at which the mtDNA 4977 IDV iszero (see Note 16).3.5. Primer-Shift PCR1. PCR amplification: Prepare a master mix containing all the components of thePCR reactions with the exception of oligonucleotide primers. For eight reactions(final volume of 50 µL) you will need the following:282.8 µL Sterile water40 µL 10× Reaction buffer40 µL 2 mM dNTPs10 µL Template DNA (single cell lysis)3.2 µL AmpliTaq ® DNA polymerase