Method for the mapping of the local AT/GC ratio along DNA

1. A method comprising:

applying specific mapping techniques and algorithms for mapping nucleotide features on a target DNA segment of length at least 1 kb base pairs to DNA segment profiles obtained through optical fluorescence images taken after performing melting analysis on a DNA segment of at least 1 kb base pairs which was stretched within a nanofluidic channel and labelled using a fluorescent dye whilst exposed to DNA denaturation conditions, wherein

the predetermined mapping techniques and algorithms comprise

time-trace rescaling the set of optical fluorescence images to generate single molecule barcode profiles by aligning the DNA segment profiles to a reference location and processing the DNA segment profiles based upon a first dilation process to filter out longitudinal thermal fluctuations in the DNA segment profiles; and

creating a consensus barcode by aligning the single molecule barcode profiles by maximizing correlation of each single molecule barcode profile with a template profile and applying a second dilation process to account for differences in the profile scaling by minimizing a difference between each DNA segment profile and a template profile.

2. The method according to claim 1 , wherein

generating the template profile comprises creating a theoretical barcode by using an extension per base pair derived from measured stretching of a DNA calibration standard of known size and an estimate of helicity and then aligning a measured single molecule barcode profile to the theoretical barcode.

3. The method according to claim 1 , wherein

minimizing a difference comprises minimizing the squared difference.

4. The method according to claim 1 , wherein at least one of:

the mapped nucleotide features relates to localized AT and GC base pair ratios;

denaturing the DNA is achieved using at least one process selected from the group consisting of:

exposing the stretched DNA segment to a predetermined temperature;

applying a chemical treatment to the stretched DNA segment; and

changing buffering conditions of the DNA segment within the nanofluidic channel.

5. The method according to claim 1 , wherein

the optical fluorescence images depict fluorescence variations along the DNA segment which are the result of either binding or unbinding of the fluorescent dye when the DNA denatures.

6. The method according to claim 1 , further comprising

driving the DNA segment into proximity of the nanofluidic channel by at least one of:

a pressure differential within the nanofluidic channel device; and

an electrical potential applied to a predetermined region of the nanofluidic channel of which the nanofluidic channel forms part.

7. The method according to claim 1 , wherein

the fluorescent dye is either an intercalating dye or a dimeric cyanine nucleic acid stain.

8. A method of creating a base pair sequence for a DNA segment of length at least 1 kb base pairs, the method comprising

applying specific mapping techniques and algorithms for mapping nucleotide features on a target DNA segment of length at least 1 kb base pairs to DNA segment profiles obtained through a set of optical fluorescence images taken after performing melting analysis on a DNA segment of at least 1 kb base pairs which was stretched within a nanofluidic channel and labelled using a fluorescent dye whilst exposed to DNA denaturation conditions, wherein

the predetermined mapping techniques and algorithms comprise

processing the set of optical fluorescence images to generate single molecule barcode profiles by aligning the DNA segment profiles to a reference location and processing the DNA segment profiles based upon a first dilation process to filter out longitudinal thermal fluctuations in the DNA segment profiles;

generating a template profile by creating a theoretical barcode using an extension per base pair derived from measured stretching of a DNA calibration standard of known size and an estimate of helicity and then aligning a measured single molecule barcode profile to the theoretical barcode;

creating a consensus barcode by aligning the single molecule barcode profiles by maximizing correlation of each single molecule barcode profile with a template profile and applying a second dilation process to account for differences in the profile scaling by minimizing squaring a difference between each DNA segment profile and a template profile.

9. The method according to claim 8 , wherein

at least one of:

the mapped nucleotide features relate to localized AT and GC base pair ratios;

denaturing the DNA is achieved by at least one of exposing the stretched DNA segment to a predetermined temperature and changing buffering conditions of the DNA segment within the nanofluidic channel; and

the DNA segment is initially driven into proximity of the nanofluidic channel by at least one of a pressure differential within the nanofluidic channel device and an electrical potential applied to a predetermined region of the nanofluidic channel of which the nanofluidic channel forms part.

10. The method according to claim 8 , wherein

the fluorescent dye is either an intercalating dye or dimeric cyanine nucleic acid stain.

11. The method according to claim 8 , wherein

the optical fluorescence images depict fluorescence variations along the DNA segment which are the result of either binding or unbinding of the fluorescent dye when the DNA denatures.