DNA Degradation

DNA Degradation

There are three factors primarily contributing to DNA degradation.

1. Contamination of bacteria and fungi (Secret nucleases and degrade DNA sample)
2. Humidity
3. Heat

If the DNA sample is dry and kept at -20 C then it is safe as microbes cannot grow here. If there is humidity then microbes will grow and enzymes will work.

Partially degrade DNA can be worked upon if fragments are like 1kb then they can be used for working.

When DNA sample is to be taken for STR or RFLP then first its quality has to be taken into account. Through previous knowledge, it is seen that STR is better as results can be obtained within hours. For RFLP large DNA without any degradation is required.

If DNA has pyrimidine dimers or is nicked / double-stranded nick then PCR analysis cannot take place.

The Quality of DNA cannot be checked from nanodrop. The best way is to run on a gel.

There is a different picture at the start of PCR and the end of PCR copy number changes.

There are thousands of copies of genomic DNA in a sample so it does not matter if it breaks from the STR site.

Hence two things have to be seen

1. Source of DNA i.e. whether it belongs to humans or others.
2. Quality of DNA i.e. intact or degraded.

Alpha satellite DNA sequence i.e. D17Z1 is specific to primates. A probe of 40 base pairs is designed for this probe will not bind in case of a sequence other than this.

So the quality of DNA determines whether one should go for RELP or remain simply limited to STR. By nanodrop or spectrophotometry, intact DNA cannot be determined. There should be enough DNA for this but in forensic DNA obtained from crime scenes is not in enough amount. The intense sensitivity of techniques has to be increased. Through agarose gel, it can be seen that DNA is intact or smear (degraded).

In quality check more DNA wastes. In this case, more appropriate is to use STR for analysis it should not be limited to 13 as there are many countries that use STR sites like 50-60. There may be chances that primer dimer will occur everywhere and product size will be small so even if DNA is degraded it would not be much of a problem. This also depends on whether we have to go for quality check or not (no quality check if DNA is less). STR analysis enables us to work with degraded DNA but it is better to have intact DNA.

The amount of DNA required for STR analysis is 1-2.5 ng. The amount should not be less than 1ng to have good results. On the other hand, the amount of DNA is more than the described range then this might result in split peaks or off-scale peaks (will go above the limit of the graph). Therefore, the standard amount should be taken to have results with a specific limit. As peak shape tells about the results.

Hence, there is no need to go above the recommended range as the results will be of no use. Thus, for this purpose, DNA has to be quantified first to see how much amount is there per microliter. Less amount of DNA will result in Allele Dropout. It is not possible for all primers to be equally efficient in multiplex PCR under some conditions, all primers may not bound equally.

Due to this, there might not be the peak of an allele. Hence it is better to use more STR sites i.e. to have results of 13 STR use 20 so that in the end-use get 13. It should also be known beforehand that which primers will perform well in a combination. More PCR tubes can be used if such a combination is not possible.

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