Internal calibration standards for electrophoretic analyses
The present teachings relate, among other things, to polynucleotide sequencing, fragment analysis and sample/lane tracking, and to polynucleotide sequencers and analyzers that employ optical detection techniques. Embodiments of the present teachings are described which include, for example, the addition of a calibration standard to a sequencing reaction. Information such as peak spacing and peak shape can be extracted from the standard.
1. A method for normalization between polynucleotide-analysis experiments using one or more electrophoresis apparatus, comprising:
(A) conducting an experiment comprising:
(i) forming a sample mixture comprising (a) a sequencing-reaction sample comprising a plurality of analyte polynucleotide fragments bearing a first set of detectable labels and (b) an internal standard comprising one or more polynucleotide fragments of known quantity bearing a fifth-dye label;
(ii) electrophoresing the sample mixture and collecting signals from the first set of labels as well as the fifth-dye label, and calculating the total of each over a selected window; and
(iii) calculating a ratio of first label-set signal to fifth-dye signal.
2. The method of claim 1 , further comprising:
(B) conducting a second experiment, comprising steps (A)(i)-(iii) using a second sequencing-reaction sample; whereby a second ratio of first label-set signal to fifth-dye signal is calculated; and
using the ratios from (A) and (B), determining a relative quantity of sequencing-reaction sample between said experiments.
3. The method of claim 2 , wherein said electrophoresis apparatus is a multi-lane electrophoresis instrument, and said experiments are carried out substantially simultaneously in different lanes of said instrument.
4. The method of claim 3 , wherein the experiment of (A) is carried out on one electrophoresis instrument and the experiment of (B) is carried out on a second electrophoresis instrument.