In this particular context, quality needs and reliability may override cost economies which might be achievable from the private sector. Further, families may be uneasy to trust such private information to private sources. Thus, for reasons of accountability and sensitivity, the Federal Government should play a large role in any large scale program of mtDNA testing of remains from Korea.

Economies of scale are important to mtDNA sequencing operations, as they are elsewhere. Due to the low volume and high cost of laboratory space and equipment in current testing operations, fixed costs are disproportionately high; thus, economies of scale are particularly striking. A centralized laboratory would be significantly more cost effective than contracting multiple small private laboratories. It is easier to oversee and control the quality of one or a few large laboratories than several smaller laboratories.

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.

VIII. RESOURCE REQUIREMENTS

DSB TOR: To determine the scientific and other resource implications of undertaking large scale mtDNA testing for identification of unassociated remains. [What are reasonable resource estimates of mtDNA sequencing identifications as currently performed? What are reasonable and likely projected cost estimates associated with performing DNA identifications for Korean War remains? What laboratory personnel are available to perform large-scale mtDNA typing operations?]

MtDNA sequencing of ancient remains is resource intensive. This is due primarily to the slow and tedious nature of mtDNA sequencing from poor source material, poor quality DNA template, amplification difficulties, and sequencing reactions that must be optimized and repeated numerous times. Furthermore, the forensic nature of evidential testing demands greater care and documentation.

Current cost estimates for DNA sequencing generally are simply not applicable to the mtDNA sequencing operations necessary for CILHI casework, because they are based on estimates of high volume sequencing operations, acceptance of low levels of base miscalls, non-forensic DNA testing standards, and on optimal DNA template. Typical costs from subsidized genome project operations are between $1 and $2 per "finished" base sequenced.

The AFDIL currently sequences 613 bases as two sets of overlapping fragments (a total of 1,046 bases), and confirms the sequence by also sequencing in the reverse direction. An openly bid contract for sequencing the same region for population studies resulted in a open market figure of nearly $1,000 per blood specimen. On the other hand, when performing testing on actual casework (including multiple bone and blood specimens), the British FSS charged the Department of Defense over $100,000 for mtDNA analysis of two cases. This is not unreasonable when considering the cost of salaries for several analysts and Ph.D. molecular biologists over a ten month period.

The DCSPER has funded the AFDIL to perform mtDNA sequencing for remains identification from Southeast Asia. Projected estimates are that 500 cases would require mtDNA testing over a five year period. A case unit for workload projections is four bones and two blood reference specimens. Current mtDNA sequencing

operations cost approximately $17,500 per case at 120 cases per year, excluding the lease cost of the facilities (approximately $9,000 per case). The largest cost components consist of a new laboratory facility, staff (primarily of one DNA Analyst and one DNA Technician per case per month), and equipment and supplies. Fixed costs are quite high for this labor-intensive equipment driven operation. Retesting accounts for a substantial proportion of the laboratory testing. Recent AFDIL casework has averaged 3.7 bone fragments tested, 5.2 extractions, 37 amplification reactions, and 55.5 sequencing reactions to obtain 1,480 bases of polished/confirmed sequence per case. In a recent AFDIL workload study, approximately 25% of the labor hours were spent in laboratory testing, 50% in data analysis, and 25% in reviewing and reporting the data (Figure 8). Full casework production operations, at ten cases per month, began in October of 1994. Due to the lack of historical data, actual operational costs have not yet been fully established, but appear to be close to projected costs. Due to the immaturity of the program, full operational efficiency has not yet been achieved.

However, the decision to perform DNA testing for unassociated remains from North Korea has yet to be made. There are over 8,100 servicemembers whose remains were not recovered and identified from the Korean conflict. The best information available indicates that no more than 3,000 of the 6,000 remains could be recovered from North Korea. However, it is anticipated that DNA testing would be performed on most cases. Preliminary returns suggest that commingling of remains will be frequent, unless perhaps, joint recovery with U.S. teams is permitted; thus the number of bones tested per case may be increased. Furthermore, a family reference database of mtDNA sequences for all families would need to be constructed for comparison purposes. The total costs for the entire operation would have to include the costs of: 1) recovery and repatriation; 2) documentation review; 3) primary identification processing by CILHI; 4) a family outreach program (Annex G); 5) the family reference mtDNA sequence database; and 6) mtDNA testing of remains.

The Korean workload could be phased in as the Southeast Asia workload ends (Figure 9).

Phase I (Annex H) of a program of DNA testing of Korean remains would require the creation of a database of family reference

mtDNA sequences. It is estimated that 40 to 70% of the 8,100 families of Korean servicemembers from the Korean conflict would be contactable and willing to provide a blood sample for this purpose. The AFDIL protocol normally requires two maternal reference specimens per family. Consequently, the creation of the family reference database is anticipated to require mtDNA sequencing of between 6,480 to 11,340 blood specimens. Some skeletal remains testing could be performed without the full generation of the family database. Blood samples can be sequenced at significantly less cost and higher production rate than ancient skeletal remains. This can be performed over a two to three year period, in current facilities, at a cost of $1.6 to $2.6 million.

Phase II (Annex I) of a program of DNA testing of Korean remains would involve the mtDNA sequencing of up to 3,000 skeletal remains. Given that one laboratory analyst with technical support can process four bone specimens per month, and an average of four bones are tested per set of skeletal remains with the marginal cost for each bone being approximately $4,400 at current efficiency and staff, the projected cost of this program is approximately $51 million in FY 98 dollars over 10 years; the annual projected cost of $4.9 million per year consists of $1.3 million in fixed costs and $3.8 million in marginal costs. Some difficult cases are far more consuming of resources than other cases. The duration of the program could be shortened or lengthened, but no less than eight years if all work is performed in the current AFDIL facility and using current technology. Recovery and preprocessing of remains by CILHI may limit the number of specimens to be tested per year.

This projection assumes a reasonably expected increase of efficiency of 50%, but efficiencies could well be much higher with the development of new technologies. Projected increases in efficiency anticipate functional and organizational shifts from case to bone sequence reporting. Improved computer assisted analysis should be in place by the time of this phase of the program; decreasing analysis time by 50% will increase throughput by 250i. The better preserved bones from Korea along with improvements in extraction and amplification could half the rate of retesting now performed in casework. One particular primer set of the four primer pairs used by the AFDIL performs substantially better than the other three, suggesting that improvements could be made to the other three systems. These

improved efficiencies are foreseeable without the development of new automated DNA technologies, which will surely come to bear within the time frames projected.

New DNA sequencing and other DNA typing technologies could achieve an order of magnitude faster turn around time for testing, and decreased testing cost. However, it will not eliminate the very labor intensive job of extraction of DNA from bone nor the analysis time.

The most speculative variable significantly affecting the cost estimate is the assumption that four bones are tested per skeletal remains. This projection does not assume all bones are DNA tested, but rather that CILHI is able to successfully segregate some skeletal remains. The number could be substantially higher given the potential for commingling.

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.

IX. NEW TECHNOLOGIES

DSB TOR: To evaluate other technologies to assist in the automation and reduction of costs associated with DNA testing. [What alternative technologies might be brought to bear that may assist current DNA identification efforts? Could new technologies improve DNA typing efforts by improving discriminatory power or enhancing recovery? Could new technologies speed DNA typing efforts? Could new technologies bring down the cost of DNA identifications? Would new technologies replace or confirm current technologies? Would new technologies permit ancillary studies to improve DNA typing efforts, e.g. assessment of original DNA template damage, quantitation of human and other DNA present, etc.? Should the U.S. military fund any investigations, research, or technology development which might enhance cost effective DNA identifications? If so, what are they?]

Advances in biotechnology are progressing rapidly, particularly as part of the Human Genome initiative. Many will undoubtedly apply to the DNA identification efforts of the U.S. military. Over the next 3 to 10 years, even without expending funds specifically to develop applicable technologies, off-the-shelf technologies are expected to permit far more rapid testing at a fraction of the cost of current testing. However, investment in technologies now will help to accelerate advances in the area of identification and particularly with respect to military efforts to identify ancient remains, such that long term cost savings would surely be realized. A relatively small investment by the military could be leveraged to achieve significant gains applicable to their needs.

Significant improvements could be achieved over the currently employed DNA typing methods and technologies. New methods and technologies could potentially improve the success of DNA typing efforts as well as substantially reduce the turn-around-time and cost of testing. Possible areas for exploration and investment include, but are not limited to, the following:

A. Specimen Preparation and DNA Extraction

The first steps in mtDNA testing of skeletal remains are sample preparation and extraction of the DNA. Significant improvements could be made in enhancing the success, speeding processing, and

making it more cost efficient.

1) Recovery Improvement

The success of mtDNA identification efforts primarily rests on the ability of the laboratory to get useful mtDNA information from the samples of skeletal remains tested. The AFDIL was successful in only half of the cases initially, but has been more successful in most recent cases from Southeast Asia. Several reextractions and re-amplifications were necessary to accomplish these successes. Not only does this demonstrate significant progress has already been made, but it also suggests that current testing is pushing the limits of technology. It is anticipated that less well-preserved remains will be returned from Southeast Asia. Early testing on Korean remains suggests that the samples will be more challenging. Even marginal improvements in mtDNA extraction will significantly impact the ability of the military to effect mtDNA identifications. Whole genome amplification through a random primer technique, use of alternative polymerases, improved buffer systems and reaction conditions are obvious directions for possible improvements.

2) Automated Specimen Preparation and Extraction

Preparation and extraction of mtDNA from bone specimens are currently labor-intensive and time-consuming processing. Even if subsequent sequencing is speeded, this step may act as a significant bottleneck. Automation of sample preparation and extraction may be possible. As already performed by some in the ancient DNA community, the surfaces of bone samples could be cleaned chemically rather than by grinding, as currently performed at the AFDIL. Then instead of mechanical breakdown of the bone samples, enzymatic dissolution of the bone may be possible, particularly when accompanied by ultrasonic agitation. Cleaned bone samples could be placed into troughs for incubation, and the dissolved extract subsequently robotically manipulated through the next stages of processing.

Automation could also apply to reference blood samples. Typing family reference samples to create a database for identification of unassociated remains will require literally thousands of mtDNA typings. Automated extraction would greatly benefit these efforts. The AFDIL currently transfers liquid blood samples to bloodstains on cards for ease of use and storage. Small punches

from the cards are then used in analyses. The development of a robotic instrument to punch bloodstain cards and then extract the card punches could dramatically speed the process.

Automation will speed processing and free labor to perform other tasks. Labor is by far the greatest cost in mtDNA typing of ancient skeletal remains. Accompanying substantial decrease in operational costs should be realized after the capitalization of the equipment. Automation may eliminate many potential manual errors in repetitive sample handling. Moreover, automation may further decrease the chance of cross contamination. The automated equipment may also be a boon to other AFDIL service casework, which has call for high volume and rapid sample processing.

3) Repair

Ancient DNA is not only degraded but damaged. Strand nicks, cross-links, extraneous molecular attachments, and other sorts of damage may prevent a polymerase from reading through the length of a DNA fragment. If the DNA reparative machinery that is normally found in a cell can be used to repair small defects in DNA target templates, then the ability to amplify such ancient DNA may be greatly enhanced. Enzymes known as ligases are commonly used in molecular biology labs to splice strands together and could repair a nick in one strand of a doublestranded fragment. A polymerase may replace a missing base by matching the complement of the opposite strand. Bacterial cloning maybe useful to repair damaged DNA using the full set of repair machinery of the host organism. Some evidence suggests that repair of the DNA before amplification can indeed assist recovery of sequence information from ancient DNA. A marginal improvement in the success of the recovery of mtDNA information from bone specimens will significantly impact current identification efforts.

B. Specimen Evaluation

Evaluation of the DNA template from which the DNA information is to be derived may permit accurate sample loading, tailored

amplification conditions, an assessment of likelihood of success, and an indication of how hard to try to recover information from the sample.

1) mtDNA Quantitation

Accurate quantitation of the sample DNA target is an important aspect of various methods of mtDNA testing. Quantitation allows accurate amounts of the sample extract to be added to an amplification reaction mixture. An absence of quantitated mtDNA should result in a determination that further testing would be an unwarranted waste of time. Quantification of source mtDNA would permit inferences of susceptibility to sampling and enzymatic errors. Unfortunately, current methods of quantitating mtDNA are not human mitochondria specific and are too insensitive to be of great value in these old skeletal remains cases. A semiquantitative sample gel electrophoresis is performed which is virtually always negative unless a significant bacterial DNA content is present. A sensitive human mitochondria specific assay could be developed to assist in this casework. Competitive PCR assays, serial dilution assays, and kinetic assays are potential methods, among others, for quantitation.

2) DNA Damage Assessment

The DNA from ancient skeletal remains is severely degraded and damaged. An assessment of the damage to the DNA to be analyzed would be valuable as an indicator of how best to scientifically approach the sample for analysis and whether or not to continue to expend time and effort in reanalysis of the sample. Such an assessment may be useful to determine whether to attempt repair of the damage before amplification. Environmental damage of DNA takes the form of certain predictable classes of chemical reaction resulting in the formation of certain specific DNA adducts. A new sensitive, rapid, inexpensive, and specific method that has recently been developed involves matrix-assisted laser desorption followed by mass spectrographic analysis.

C. DNA Amplification

Any mtDNA analysis of skeletal remains will require amplification of the few mtDNA template strands present. This is a critical step in the mtDNA testing. The basic PCR amplification process has not changed substantially since it was first introduced.

1) Alternative Polymerases

The PCR reaction involves the polymerase enzyme produced from a microbe which lives in waters of hot springs called Thermus Aquaticus and known as the Taq polymerase. While Taq polymerase is an excellent polymerase for most DNA amplifications, it does have its limitations. The knowledge of DNA polymerases has recently increased and continues to improve. Newer alternative polymerases offer the possibility of greater fidelity, processivity, and ability to process damaged template or tolerate Taq inhibitors.

2) Inhibition

Inhibition of the PCR amplification reaction is often encountered in the processing of old skeletal remains. Currently, an enormous effort is expended in overcoming a variety of technical hurdles encountered with AFDIL cases. Improvements in overcoming inhibition are likely through better optimization of reaction conditions, better DNA extraction methods, and through alternative polymerases, as previously mentioned. Because inhibition directly prevents the success of DNA typing, even marginal increases are likely to be significant.

D. DNA Analysis

Currently mtDNA analysis has been performed using conventional sequencing on conventional equipment. Due to the Human Genome Initiative advances in DNA analysis are in rapid development. Several possibilities exist that could dramatically increase throughput and decrease the cost mtDNA analysis. Complete automation with integration of sample preparation, DNA