Kits and methods for diagnosing lung cancer

A method for measuring methylation ratio of DNA from a human subject suspected of having lung cancer, the method comprising:

• (a) subjecting DNA from a sample obtained from the subject to digestion with at least one methylation-sensitive restriction endonuclease, thereby obtaining restriction endonuclease-treated DNA;
• (b) co-amplifying from the restriction endonuclease-treated DNA at least one restriction locus having increased methylation in lung cancer DNA compared to normal non-cancerous DNA and a control locus, wherein the at least one restriction locus is selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 3, SEQ ID NO:
• 4, SEQ ID NO:
• 5, and SEQ ID NO:
• 6, thereby generating an amplification product for each locus;
• (c) determining a signal intensity for each generated amplification product;
• and
• (d) calculating a ratio between the signal intensities of the amplification products of each of said at least one restriction locus and the control locus, thereby measuring methylation ratio of DNA from the human subject.

A kit for identification of lung cancer in a human subject, the kit comprising:

• at least one methylation-sensitive restriction enzyme for digesting DNA from a sample from a human subject;
• and
• a plurality of primer pairs for co-amplification of at least one restriction locus and a control locus following digestion with the at least one methylation-sensitive restriction enzyme, wherein the at least one restriction locus is selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 3, SEQ ID NO:
• 4, SEQ ID NO:
• 5, and SEQ ID NO:
• 6.

A system for identifying lung cancer in a human subject, the system comprising:

• (i) at least one methylation-sensitive restriction endonuclease for digesting DNA from a sample from a human subject;
• (ii) a plurality of primer pairs for co-amplification of at least one restriction locus and at least one control locus following digestion with the at least one methylation-sensitive restriction enzyme, wherein the at least one restriction locus is selected from the group consisting of SEQ ID NO:
• 1, SEQ ID NO:
• 2, SEQ ID NO:
• 3, SEQ ID NO:
• 4, SEQ ID NO:
• 5, and SEQ ID NO:
• 6;
• and
• (iii) computer software stored on a computer readable medium, the computer software directs a computer processor to perform the following steps:
• determining signal intensities for the at least one restriction locus and the control locus following their amplification;
• calculating signal ratios between the signal intensities of each of the at least one restriction locus and the control locus;
• comparing the calculated signal ratios to at least one reference ratio;
• and based on the comparison, outputting whether the DNA sample is lung cancer DNA or healthy DNA.