Chem 8157

I have the following questions and comments for Greg and the rest of the class:

1. Data Analysis - We need more detail on the limit of detection of the MCE analysis and for the sequencing reaction? Express the results in terms of base paris. How does the amplicon size affects these limits?

2. Data Analysis - According to the paper (page 5103, before section 2.5), the quantification accuracy is 30%, the sizing resolution is 5%, and the sizing accuracy is 10%. How do these figures of merit limit the types of haplotypes that can be analyzed? For example: what is the minimum size difference? How much dog hair can the sample tolerate?

1. For the MCE analysis, the paper states that detection limits were from 60 to 30 copies of mtDNA, corresponding to 1-0.4 ng/uL of amplicon. These segments each contained about 400 bp, so this corresponds to a total detection limit of about 12000 bp.

For the sequencing reactions, the paper states that detection limits were ~500 mtDNA copies for amplicons that were 400 nucleotides in length and 250-125 mtDNA copies for amplicons that were 100 nucleotides in length. This corresponds to about 200000 total bp in the first case and 25000 bp in the second case. Therefore, the detection limits get better as we select a sequence to amplify.

2. The sizing resolution limits somewhat the type of haplotypes that can be analyzed. For example, if we are looking at a region that is 163 bp long (this is the region of Cyt-b that the human-specific primers used in the article amplify), then we must not have any interfering amplicons that are in the range of 154-171 bp. In this particular experiment, the amplicons produced by the universal primers are at 130 bp (for the dog) and 116 bp (for the human) - far enough away to resolve.

I'm not sure I understand your question about the amount of dog hair that can be tolerated. It seems like the method is designed to amplify mtDNA from single hair shafts (one at a time) to determine species. As far as I can tell, PCR would be done on each individual sample of interest (with the universal and human specific primers present) and then the amplicons would be analyzed by MCE to determine the presence of the Cyt-b amplicon produced only by the human-specific primer.

Regarding dog hair, i agree that my question was not clear. I meant, "if dog hair is a contaminant, how much DNA from dog hair can be tolerated in the analysis of a human DNA sample? Consider that according to Matsuda, the human primers are not 100% specific (see page 5107).

I still couldn't figure it out - I feel that the article doesn't present enough information about the extent of contamination. It mentions that "faint MCE peaks (~0.5 ng/uL) were observed from some animal samples with high mtDNA content" on page 5107, but does not mention either the location of the peaks in the chromatogram or what exactly constitutes "high" mtDNA content. Would these peaks overlap with the ones produced by human DNA? Since the only false positives were seen with animal blood samples, would hair samples contain enough mtDNA to produce similar false positives?