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Method of determining biological/molecular age

a biological/molecular age and biological measurement technology, applied in the direction of microbiological testing/measurement, biochemistry apparatus and processes, etc., can solve the problems of aging and death, increased susceptibility to disease and ultimately death, and reduced energy generation capacity

Inactive Publication Date: 2005-11-17
SEIDMAN MICHAEL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aging is a complex process that involves metabolic and physiologic changes that lead to an increasing susceptibility to disease and ultimately death.
The mitochondrial DNA damage leads to reduced capacity for energy generation within the mitochondria and ultimately causes aging and death.
It is not difficult to comprehend that if you remove approximately one-third of the mitochondrial DNA you will have significant problems with energy generation.
It has been found that even minor amounts of this deletion severely alter energy production and cellular function.
It is known that certain tissues are more susceptible to oxidative damage (damage from free radicals) and reduced energy supply.
For example, brain, eye, inner ear, and all muscle can accumulate high amounts of these deletions and they become more susceptible to free radical damage than other tissues.
Furthermore, caution must be exercised when these genes are manipulated, because of the potential to trigger cancerous change.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Protocol

Detection and Quantification of MtDNA Deletion (MtDNA4977)

[0031] A detailed protocol is found in reference 35, N—W Soong and N. Arnheim, Meth Enzymol., 421-431, 1996.

[0032] Primers (Designed in our Laboratory):

(SEQ ID NO:1)Mt1C:AGG CGC TAT CAC CAC TCT TGT TCG(13,176-13198)(SEQ ID NO:2)XMt2:AAC CTG TGA GGA AAG GTA TTC CTG C(13,501-13,477)(SEQ ID NO:3)Mt1A:GAA TTC CCC TAA AAA TCT TTG AAA T(8224-8247)

[0033] Primers are end-labeled with (γ-32P) ATP using T4 Polynucleotide Kinase. Unincorporated nucleotides are removed by spinning through P4 columns. These primer lots are prepared to give approximately 10× concentration for PCR (5 micromolar) and are diluted directly into the PCR mix.

example 2

PCR Analysis:

[0034] PCR is carried out in 50 microliter volumes in 1×PCR buffer, containing 1.5 mM MgCl2.

[0035]32P end-labeled primer concentration is 0.5 micromolar. [0036] Deoxy-nucleoside triphosphate (dNTPs) 200 micromolar. [0037] 2.5 Units of Taq polymerase. [0038] 100-1000 ng of genomic DNA. [0039] Primers for Total MtDNA: Mt1C and Mt2, fragment size 324 bp. [0040] Primers for Deletion: Mt1A and Mt2, fragment size 303 bp.

[0041] Cycle Parameters:

Initial denaturation at 94° C. for 3 min.Denaturation at 94° C. for 30 sec.----- \Annealing at 54° C. for 30 sec30 cyclesExtension at 72° C. for 1 min.----- / Followed by 7 min extension at 72° C.

[0042] PCR conditions are identical for total and deletion-specific reactions except that deletion-specific reactions are run for 30 cycles and control PCR is carried out for 15 cycles.

example 3

Polyacrylamide Gel Electrophoresis

[0043] After PCR, 10% (5 microliters) of each reaction is electrophoresed through 8% polyacrylamide gel. The gel is dried and counts from each specific band are quantitated with a PhosphorImager (Biorad) after 15-24 hr exposure.

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Abstract

Methods of obtaining a measurement indicative of oxidative stress and the molecular age of an individual include the step of detecting a mitochondrial DNA deletion and correlating the quantity of the deletion with a measurement of a parameter related to oxygen metabolism.

Description

RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 10 / 271,469 filed Oct. 15, 2002, which is a continuation-in-part of U.S. patent application Ser. No. 09 / 885,732 filed Jun. 20, 2001, which claims priority of U.S. Provisional Patent Application Ser. No. 60 / 212,747 filed Jun. 20, 2000.SEQUENCE LISTING [0002] This application contains a Sequence Listing that is being submitted herewith as a separate document. FIELD OF THE INVENTION [0003] The present invention relates to physiological measurements indicative of fitness and aging. In particular, the invention relates to a method of obtaining a measurement indicative of oxidative stress in an individual. This invention relates to a method of determining the biological / molecular age of a human and more particularly to such a method which involves detection of and quantification of aging deletions and comparison of the quantified deletions with known normative data. BACKGROUND OF THE INVENTIO...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q1/686C12Q1/6883C12Q2600/156C12Q2545/114
Inventor SEIDMAN, MICHAEL
Owner SEIDMAN MICHAEL
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