PCR based diagnosis in the presence of 8% (v/v) blood

While Taq polymerase was totally inhibited by the presence of as little as 1 mu l of blood, Tth polymerase, from Thermus thermophilus HB8 was able to successfully amplify a specific 1.3 kb target sequence in PCR reaction mix containing up to 4% (v/v) blood. Clearly there are no specific inhibitors of DNA polymerase activity in whole blood. A further increase in the amount of blood inhibited the ability of Tth polymerase to amplify DNA. It is believed that this loss of DNA polymerase activity is due to the entrapment of target DNA by the large amount of coagulated organic material present in the blood containing PCR samples. Thus the target DNA is inaccessible to the DNA polymerase. The addition of one unit of the single stranded DNA binding protein, T4 genes 32 protein, further increases the accessibility of Tth polymerase to the target DNA.