DNA Quantification
Using spectrophotometry/fluorimetry
Spectrophotometry
o Absorbance (A) = Optical Density (OD)
o Measure DNA at 230/260/280 nm
o 1 OD = 50 ng/uL dsDNA | 40 ng/uL RNA | 33 ng/uL oligonucleotides
o 260 nm Nucleic acids naturally absorb light at 260 nm due to phenolic ring
o No information about DNA degradation
o Poor analytical specificity
o Other substances can absorb at measured wavelengths
UHMW DNA Sample at 3 points, calculate mean & coefficient of variation [(SD/mean) x 100]; CoV
must be < 30% for conc. readings to be accurate
Fluorometry
o Fluorescent spectrophotometry
o Fluorescent dye binds to AT residues in minor grooves
o Dye does not bind to proteins, RNA or single stranded DNA
o Fluorescent level is low until dye binds molecule of interest
o Requires control sample with similar GC content as experimental sample
o Can combine fluorometry with spectrophotometry
o Use in NGS prep
o DNA conc. of the library must be accurately determined,
Too high Increased risk individual templates will be amplified as mixture;
NGS data cannot be interpreted
Too low Poor data yield
Quality Assessment of DNA
o A260/280 1.8 – 2.0 = pure DNA; Above 2 = RNA contamination; Below 1.8 = Protein
contamination
o A260/230 2.0 – 2.2 = pure DNA
o Use of RE can be part of the QC; confirms DNA is present & extent of degradation
Use of RE to detect a mutation in PCR product
o Only possible if RE site is created/lost at the mutation site
o PCR-RFLP (PCR – Restriction Fragment Length Polymorphism)
o PCR product is digested with RE prior to running on gel
Components of a RE Digest
o Core components DNA, buffer, enzyme
o Successful RE digest requires correct pH, ionic strength, temperature
o DNA must be dsDNA
o Can use digest pattern to determine location of RE cut sites
o Need to quantify DNA conc. first
o Don’t mix buffer & enzymes from diff. companies
o 1 RE unit = The amount of enzyme required to digest 1 ug of DNA in 1 hour
o Keep RE on ice; add RE last; Use excess RE to compensate for DNA inhibitors + liquid handling
Using spectrophotometry/fluorimetry
Spectrophotometry
o Absorbance (A) = Optical Density (OD)
o Measure DNA at 230/260/280 nm
o 1 OD = 50 ng/uL dsDNA | 40 ng/uL RNA | 33 ng/uL oligonucleotides
o 260 nm Nucleic acids naturally absorb light at 260 nm due to phenolic ring
o No information about DNA degradation
o Poor analytical specificity
o Other substances can absorb at measured wavelengths
UHMW DNA Sample at 3 points, calculate mean & coefficient of variation [(SD/mean) x 100]; CoV
must be < 30% for conc. readings to be accurate
Fluorometry
o Fluorescent spectrophotometry
o Fluorescent dye binds to AT residues in minor grooves
o Dye does not bind to proteins, RNA or single stranded DNA
o Fluorescent level is low until dye binds molecule of interest
o Requires control sample with similar GC content as experimental sample
o Can combine fluorometry with spectrophotometry
o Use in NGS prep
o DNA conc. of the library must be accurately determined,
Too high Increased risk individual templates will be amplified as mixture;
NGS data cannot be interpreted
Too low Poor data yield
Quality Assessment of DNA
o A260/280 1.8 – 2.0 = pure DNA; Above 2 = RNA contamination; Below 1.8 = Protein
contamination
o A260/230 2.0 – 2.2 = pure DNA
o Use of RE can be part of the QC; confirms DNA is present & extent of degradation
Use of RE to detect a mutation in PCR product
o Only possible if RE site is created/lost at the mutation site
o PCR-RFLP (PCR – Restriction Fragment Length Polymorphism)
o PCR product is digested with RE prior to running on gel
Components of a RE Digest
o Core components DNA, buffer, enzyme
o Successful RE digest requires correct pH, ionic strength, temperature
o DNA must be dsDNA
o Can use digest pattern to determine location of RE cut sites
o Need to quantify DNA conc. first
o Don’t mix buffer & enzymes from diff. companies
o 1 RE unit = The amount of enzyme required to digest 1 ug of DNA in 1 hour
o Keep RE on ice; add RE last; Use excess RE to compensate for DNA inhibitors + liquid handling
