1) Automation of Sequencing Reactions

Current sequencing reactions are performed manually. Automated methods for performing sequencing reactions are available. Automation not only can achieve higher throughput and reduce overall costs, but also may achieve better results. The large number of steps involved in current testing creates the potential for manual errors to be made. Automation will allow dye primer and T4-sequenase chemistries that are too tedious for manual production sequencing casework efforts.

2) Capillary Electrophoretic Arrays

Current sequencing technology is based on gel electrophoresis in which DNA is pulled across a slab of polyacrylamide gel using an electric current. The speed of this system is determined by the electric voltage applied, but the voltage is limited by the heat generated by the current in the gel. If the electrophoresis is carried out in a very fine capillary then a substantially higher voltage can be applied and a faster run time achieved. Run times are measured in minutes instead of hours. Due to the higher resolution, a greater number of bases can be read per run. Furthermore, a capillary can be sample loaded in an automated fashion, obviating the need for pouring and loading a gel. As a result, many DNA samples (up to 36 or 48) can be run simultaneously on a slab gel, but only one DNA sample can be loaded in a capillary per run. Instruments that employ arrays of capillaries are currently in development and could dramatically speed DNA sequencing using conventional sequencing chemistries, with an accompanying reduction in cost.

3) Sequencing Microchips

Another major new technology under development is sequencing by hybridization (SBH) performed on microchips. Microlithography is an engineering tool used in the manufacture of computer microchips. Microlithography will permit the cost-effective generation of arrays of thousands of DNA probes which enables SBH technology. One of the first prototype "DNA chips" will sequence the D-Loop of mitochondria. This microchip can perform sequencing at a fraction of the time and cost of current DNA

sequencing methods. These microchips should be first used to confirm current sequencing technology and perhaps later employed as the primary method of sequence information.

4) Single-Point Polymorphism Assays

An alternative technique to sequencing which captures most sequence data, developed by Dr. Mark Stoneking in conjunction with Dr. Henry Erlich's laboratory, uses hybridization of sequence specific oligonucleotide (SSO) probes to amplified segments of HVl and HV2 that have been immobilized on a membrane. These SSO probes are designed to detect variation at specific polymorphic nucleotide positions to produce a set series of yes/no results known collectively as a "mitotype". This system is more limited in its discriminatory power than full sequencing; but it offers the advantages of being quick, inexpensive, and does not require great technical skills. Preliminary results suggest that it is more sensitive than current sequencing methods. An analysis of mtDNA SSO-type variation in 142 U.S. whites using 23 SSO-probes revealed 99 different types, and an average probability of identity of 1.90%. A commercial dot/blot strip is under development for the typing of the mitochondrial D-Loop. Moreover, a similar method could be used to interrogate sites outside the D-Loop which are so spread out as to make the cost of sequencing for these polymorphisms prohibitive.

Dot/blot technology could be reduced to microchip probe arrays. Site polymorphisms could also be interrogated through the use of oligonucleotide ligation assays and detection systems, which offer the advantages of greater specificity and large scale multiplexing. These various systems for detecting point polymorphisms are highly amenable to automation. Also denaturing gel electrophoresis could be used as a method to screen for polymorphisms. These technologies could be employed as a screening tool to sort bones with cases, as a method of confirming sequencing results as well as a primary mtDNA typing method to increase the discriminatory potential.

5) Computer Software

Currently, far greater time is spent in the analysis of the sequencing results than in sequencing itself. A significant portion of the time is spent appropriately formatting the data and then checking the raw sequencing data and reading. Computer

software could be developed which could perform these functions more quickly, more accurately, and without tedium. A preliminary neural network system already demonstrates a several fold diminution in the rate of instrumental miscalls from the raw output of the Applied Biosystems, Incorporated, model 373a instrument currently used by the AFDIL.

A laboratory information management system (LIMS) could be developed which could greatly improve the efficiency of current efforts. A LIMS could integrate robotic systems, capture instrument data and export it into analysis software, generate reports, improve case tracking and perform quality assurance functions.

The idea that new technologies should be developed and explored, should not be construed as implying current technologies are in any way inaccurate or not worthwhile. Current technology is, however, costly and time consuming, and mitigation of these burdens would be welcome.

The Task Force finds that new technologies should be surveyed for the best prospects for increasing the success and efficiency of DNA identification of ancient skeletal remains. Future investments should be guided by the progress of technology and appropriate advice.

X. SCIENTIFIC BASIS

DSB TOR: To determine the degree of scientific experience and expertise available to support the Task Force findings. [Is the current scientific foundation for DSB findings to these questions adequate or is it premature to answer the foregoing questions? is further investigation and experience necessary or helpful in determining the answers to the above questions? Should the Task Force be reconvened or should the military otherwise revisit the above issues in the future?]

The foregoing Task Force Report findings have been based on substantial scientific evidence. While it is always true that more information can and will be produced bearing on important scientific questions, these findings do not seem premature. We note that a Quality Assurance Oversight Committee has been created by direction of the ASD (HA), composed of civilian consultants, which will act to ensure that efforts will continue to be performed with the greatest scientific integrity.

The Task Force supports the ASD (HA) in the creation of a scientific advisory board composed of civilian technical consultants.

The Task Force finds that current DNA identification efforts are supported by sufficient scientific evidence to proceed, in particular with application of mtDNA sequencing to ancient remains from the Korean conflict.

The Defense Science Board Task Force find that ancient skeletal remains can be accurately and successfully identified through mtDNA testing. The current methods of mtDNA sequencing are scientifically sound and can be performed in a reliable manner. Furthermore, mtDNA can be used to identify unassociated remains through the creation of a database of family reference specimens.

1. FEASIBILITY:

The Task Force finds that identification of so-called ancient skeletal remains by a program of mtDNA testing is possible, particularly in association with other information. A few specimens may remain unresolved. Although contenders may emerge; at this time, mtDNA sequencing technology is the most appropriate technology.

2. FACTORS:

The Task Force finds that the present probability of coincidental matches between mtDNA control region sequences is no more than a few percent. Once sequences from 500 members of a population have been determined, precise statements about the chance of a false association of a set of remains with a family will be able to be made. Published data may be of value, but samples will be needed from Southeast Asian populations. The precision is expected to suffice in the vast majority of cases, given other non-DNA evidence, to effect DNA identification of unassociated Korean remains. It will not be possible, however, to identify every bone. A great deal can be done with anatomical and historical evidence alone.

The Task Force finds that control of contamination is essential to PCR-based laboratory testing. Some contamination is unavoidable, particularly in mtDNA testing of ancient remains, but it does not preclude reliable casework testing where redundancy, good laboratory practices, and appropriate cautionary language are used and constant oversight is maintained.

The Task Force finds that casework experience demonstrates capability to type Korean skeletal remains.

3. RELIABILITY:

The Task Force finds that appropriate measures must be taken to prevent and control possible contamination in the testing laboratory.

The Task Force finds that current AFDIL protocols, if diligently performed, are capable of generating quality mtDNA sequence identifications.

The Task Force finds that adequate quality assurance requires accreditation and an oversight board.

The Task Force finds that the program for mtDNA sequencing quality assurance promulgated by the Office of the Assistant Secretary of Defense (Health Affairs) for identification of ancient remains is adequate and responsive.

4. OTHER DNA TARGETS: The Task Force recommends that the AFDIL investigate the potential to perform DNA typing outside the mtDNA control region.

5. FAMILY REFERENCE DATABASE: The Task Force finds that, with a reasonable effort, a sufficient proportion of families are expected to provide DNA samples to allow identification of many of the unassociated remains from Korea through mtDNA testing, and to attach meaningful probability statements in those cases.

The Task Force recommends consideration be given to the collection of DNA reference specimens from maternal and paternal family members in case future technology permits nuclear DNA testing. Collections from nonmaternal kindred members (e.g. children) should be made with full disclosure of realistic expectations.

6. STATISTICAL DATABASE:

The Task Force finds that the existing databases, and those anticipated from the family collections, provide an adequate basis for the current mtDNA sequencing efforts. The Task Force recommends an expansion of the current AFDIL

database.

7. LARGE-SCALE OPERATIONS:

The Task Force finds that current mtDNA testing efforts could be augmented for large scale operations. There are strong arguments for a centralization of the laboratory work for the sake of vigorous oversight, quality control, and accountability.

8. RESOURCE REQUIREMENTS:

The Task Force finds that current mtDNA testing efforts at AFDIL are funded appropriately for the Southeast Asia mission. The Task Force concurs with the projections that analysis of Korean War remains could be accomplished over the next twelve years with an increase of funding of approximately $2 million per year over the cost of current operations.

9. NEW TECHNOLOGIES:

The Task Force finds that new technologies should be surveyed for the best prospects for increasing the success and efficiency of DNA identification of "ancient skeletal remains. Future investments should be guided by the progress of technology and appropriate advice.

10. SCIENTIFIC BASIS:

The Task Force supports the Assistant Secretary of Defense (Health Affairs) in the creation of a scientific advisory board composed of civilian technical consultants.

The Task Force finds that current DNA identification efforts are supported by sufficient scientific evidence to proceed, in particular with application of mtDNA sequencing to ancient remains from the Korean conflict.

The Task Force notes that the technology for mtDNA sequencing will continue to improve and become less costly due to activities both within and outside the military. Military efforts to identify skeletal remains using DNA will greatly impact law enforcement and contribute to the science of molecular biology, and molecular anthropology.