DNA Quantification

Pico green Microtitre Plate Assay
Pico green is a dye that binds to double-stranded DNA. It is more sensitive than ethidium bromide or cyber green. Gives fluorescence segment is a result of binding. There will be 96 wells, 80 will be test samples and 16 will be calibrations of a known amount of sample. Within 30 minutes the amount of DNA in 80 samples can be seen. 5 Ul of samples 195 Ul of pico green solution. Then examine it in a fluorometer ( measures fluorescence ). The amount of calibrations ‘ DNA is known. The signals are then compared with that of the unknown. A standard curve of calibrations can be made.

Single-stranded DNA cannot be quantified as the dye is specific for double-stranded DNA only. This method has high sensitivity but when compared with slot blot it has a drawback in case of slot blot it is known hot whether DNA belongs to humans or not ( thought 40 bp probe ). But in the case of pico green, fluorescence will come in all cases. It is not primate-specific. Through it, any DNA can be quantified.

Aliquant: Human DNA Quantification Method

There are alu sequences in our genome those which are cut restriction endonuclease alu. Repetitive alu sequences. The length of these alu sequences is about 30 bp and the number is more than 1 million. They make 10 % of our genome. They are transposable elements. One set of primes is required only. If there are one or two copies of the genome then it will be enough as it will have 1 million copies each enough amount of alu. If the standard curve is made with known amounts of genome then it can be seen that what is the amount in a sample the number in the product will be specific. If there is one genome target copy then after 25 cycles of PCR. the number of copies will be 8388608. A number already more than 1 million thus through known amounts we will make a standard curve e.g if

One copy ➡️ 1ng
Two copies ➡️ 2ng

Real-Time PCR

In Real-Time PCR it can be quantified that what is the starting number template quantity in sample. Sensitivity will increase here, micrograms can be measured here and it can be determined how much genome copies are there in our sample.

End Point PCR

Either we can take some STR sites or all sequences to be amplified in PCR. By taking known amounts in the form of dilutions a standard curve can be made. It is a cheap method, just like a common PCR. Syber green can be used as its sensitivity is more than ethidium bromide. (Syber green is added after reaction). Some number of cycles will be carried for both known and unknown. Thus the amount of DNA in a sample can be seen.

Advantages

If there are some PCR inhibitors then we can have knowledge about them monitor them.

It is a cheap method as the cyber green used in it is cheap and is more sensitive than ethidium bromide.

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DNA Quantification | Human DNA Quantification Method | Advantages