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F. Samuel Baechtel

FBI Laboratory
Quantico, Virginia

The intent of this document is to give the must be free of substancesthat would compromise
reader an overview of the processes of recovery, restriction endonuclease activity; and the quantity
cleanup and quantiflrcation of DNA as they are ap- of DNA retrieved must be sufltcient to meet the
plied to body fluid stains analyzed in the crime lab- analytical requirements of the test procedure.
Factors Affecting the Size of Recovered DNA
There are several factors that can affect the
Diploid human cells each contain about 6 pg molecular size of the DNA recovered from biologi-
of DNA, contained almost totally in the 46 chro- cal evidentiary specimens. One of the most
mosomes. About lVo of the total cellular DNA is common causes of DNA destruction is the action
located in the mitochondria. Haploid cells (sper- of the endo- and exonucleasesthat are ubiquitous in
matozoa and ova) contain one half the diploid nature. While the analyst can do nothing to pre-
quantity of DNA, or about 3 pg/cell. For human vent the destructive action of nucleases prior to
somatic cells, only the erythrocytes are devoid of specimen collection, there are several steps that can
chromosomal DNA. be taken to abrogate their activity once the speci-
Since crime scene evidentiary materials most men environment can be controlled. Specimens
often are shed body fluids, it is useful to estimate should be maintained cool and dry prior to the
the quantities of DNA expected to be in common commencement of DNA recovery procedures. The
fluids. For example, the normal number of leuko- extraction solution should contain a metal chelator,
cytes in human peripheral blood generally ranges such as ethylenediaminetetraacetic acid (EDTA),
between 5 and l0 million cells per ml of blood.
to prevent the activity of DNases. Moreover, pi-
This equates to DNA concentrations that can vary pette tips, glassware, and appropriate reagent solu-
between 30 y"g/ml and 60 pg/ml of peripheral
tions should be autoclaved before use to inactivate
blood. The average number of spermatozoa per ml
any nuclease activity that might be present.
of semen is 150 x 106/ml (Mann and Lutwak-Mann
A common abuse inflicted on DNA in the lab-
1981). Thus the DNA concentration in semen is
oratory during recovery is excessive shear force,
considerably higher than in blood and averages
such as prolonged agitation on a vortex mixer.
about 450 pg/ml. Each ml of semen can contain
Such mishandling can result in DNA fragmentation
about 5 x 106 leukocytes which contribute another
to an extent that the molecular weights of the frag-
30 pg DNA per ml. Thus semen from an average
ments are too low for the remaining steps in the
male possessesapproximately 480 pg DNA per ml.
Considering that a satisfactory autoradiogram RFLP procedure to be carried out.
image of a restriction fragment linked polymor- The molecular weight of intact DNA in the
phism (RFLP) pattern can be obtained from 20-50 cell nucleus is about 1011daltons. Careful recovery
ng of human genomic DNA, one can calculate of DNA that has not been severely affected by nu-
that, with a lOOToyield, the DNA would have to clease activity or shear forces should be of molecu-
be recovered from 4.000-10.000 nucleated cells at a lar weight about 107 daltons (Gross-Bellard et al.
minimum. Since the yield of DNA from stain mate- r973).
rial routinely is less than lOO7o,more than the min-
Factors Affecting the Susceptibility of DNA to
imum number of nucleated cells is required to give
Restriction Digestion
a satisfactory RFLP pattern.
The recovery process must not only avoid de-
GOALS OF THE DNA RECOVERY PROCESS struction of the DNA, but it must render the DNA
There are three major goals that must be met suitable for digestion by restriction endonuclease
during the DNA recovery processes if the RFLP (RE) activity. Two factors can reduce the ability
typing analysis is to be successful. The recovered of RE to fully digest DNA: the presence of histone
DNA must be of high molecular weight; the DNA proteins that remain attached to the DNA and pre-

vent access of RE to all possible restriction sites; would destroy the DNA. Many procedures call for
and the presence of adventitious substancesthat are an overnight extraction period (Kanter et al. 1986;
inhibitory to the catalytic activity of the RE. Gill et al. 1987).
Histones are removed from the DNA and de-
natured by the action of detergents. This is usually Denaturation,/Hydrolysis of Proteins
done at moderately elevated temperature (for ex- The presence of detergents in the stain extrac-
ample, 56' C). The denatured histones then are hy- tion buffer is responsible for the lysis of cellular
drolyzed to peptides and amino acids by the action
membranes and for the dissociation and denatura-
of a proteolytic enzyme incorporated into the ex-
tion of the histone proteins Ihat are tightly attached
traction solution.
to the DNA strands. Detergents destroy the sec-
Adventitious substancesthat are present in the
ondary and tertiary structures of proteins which
DNA preparation can inhibit the catalytic activity
leads to their decreased solubility in aqueous solu-
of the RE. Such adventitious substances either
were present on the surface upon which the body tion and increased susceptibility to the hydrolytic
fluid was deposited (for example, dirt, salt, acids) activity of proteolytic enzymes. A commonly em-
or were introduced during the DNA recovery (for ployed detergent is sodium dodecylsulfate (SDS).
example, phenol, chloroform). Regardless of their Proteinase K (F'beling et al. 1974) is widely
source, contaminants can be removed by precipita- used in procedures for the isolation of DNA as an
tion of the DNA from solution with ethyl alcohol, effective tool for the hydrolysis of histone proteins.
leaving the contaminants in solution; by dialysis; or This enzyme is active across a wide range of pH, is
by filtration. active in the presence of SDS (in fact, its activity is
enhanced), and it is unaffected by metal chelators
Factors Affecting the Quantity of DNA Recovered
such as EDTA.
The quantity of DNA recoverable from foren-
sic specimens is influenced by a number of factors. Removal of Denaturation Products
Obviously, the size of the stain or tissue sample af- Denatured proteins can be removed effectively
fects the potential quantity of DNA that is avail- from the extraction solution by treatment with
able for recovery. In addition, the type of surface phenol and chloroform. Phenol, and to some extent
onto which the fluid has been deposited can dra- chloroform, is an effective protein denaturant.
matically affect the level of DNA recovery for
Moreover, the products of denaturation and pro-
body fluid stains.
teolysis are soluble in phenol (Kirby 1957). Some
GENERAL METHODS FOR RECOVERY AND recovery procedures call for the use of phenol and
EVALUATION OF DNA chloroform mixtures, while others use phenol first
followed by one or more treatments with chloro-
The process of recovering DNA from forensic
form to ensure complete removal of phenol. Isoa-
specimens can be broken into five steps: (l) Rehy-
myl alcohol is included in mixtures of phenol and
dration of the stain and solubilization of the stain
chloroform to reduce the tendency of proteins to
components; (2) denaturation and hydrolysis of
foam when they are denatured during shaking with
proteins; (3) removal of denatured proteins; (4) pu-
the organic solvents (Marmur 1961).
rification of the DNA; and (5) quantification of
DNA and assessmentof its quality for RFLP anal- Purification of DNA
ysis. Each of these steps will be considered further.
Additional cleansing of the DNA is necessary
Solubilization of Stain Components before restriction digestion is attempted. The pur-
Dried stains can lose considerable water in the pose of this step in the procedure is to remove
course of drying. This water must be replaced and small molecules that are potential inhibitors of RE
the stain components resolubilized for the recovery catalytic activity. Three approaches to additional
procedures to succeed. During this phase of the re- DNA cleanup that have been used are: (l) Precipi-
covery process, the DNA must be protected from tation of the DNA from solution by ethanol; (2) di-
unnecessary degradation. Stains are cut and solubi- alysis of the DNA solution against large volumes
lization is accomplished by soaking the stain in of buffer (Kanter et al. 1986); and (3) ultrafiltration
buffer, usually at temperatures from 37' C to 56" C. through selectively permeable membranes (Marashi
A chelator of magnesium, such as EDTA, is in- et al. 1985\.
cluded to prevent the action of the nucleases that

Assessment of Quantity and Quality of DNA readily determined by yield gel procedures. Yield
gels are small agarose gels that contain ethidium
There are four approaches that can be used to bromide. A fraction of each DNA specimen along
determine the quantity or quality of DNA. While with DNA calibration standards are subjected to a
all four permit quantifrcation of DNA, not all brief electrophoretic separation at relatively high
enable a comprehensive assessmentof DNA qual- voltage. The standards cover the range from 15 ng
itv. DNA up to about 300 ng DNA, generally in dou-
Ultraviolet Absorption bling steps. After the electrophoretic run is com-
plete, the gel is placed under UV light and a pho-
The purine and pyrimidine bases in DNA tograph is taken. For analyst safety, test gels
absorb light in the ultraviolet (UV) region of the should not be evaluated while the gel is irradiated
light spectrum. While each of the four bases that with UV light. The intensity of fluorescence of the
occur in DNA has its own specifrc absorption max- DNA test specimen(s) in the photograph is com-
imum, the composite maximum occurs at 260 nm. pared with the intensity of the standards and an es-
A DNA solution of concentration 50 p,g/ml will timate is made of the DNA concentration in the
yield an absorbency at 260 nm that is equal to 1.0. test specimen(s). This method of determining DNA
The lower limit of detection for DNA by UV is concentration in test specimens is semi-quantitative
about 0.5 pg DNA,zml. and is the least accurate of the available quantita-
Ultraviolet measurements are useful also for tive methods.
determining if proteins and/or phenol remain with Yield gel measurement is the most rapid
the DNA after isolation and purihcation. If pro- method of assessing the molecular weight of the
teins have been effectively removed from the DNA in a test specimen. High molecular weight
DNA, the ratio of absorbencies determined at 260 DNA remains as a compact band that does not mi-
nm and 280 nm will equal about 1.8. Likewise, grate far from the origin during the brief electro-
when the ratio of absorbencies 260 nm/270 nm phoretic period. In contrast, DNA that has been
equals 1.2, the preparation can be considered to be degraded will migrate more rapidly than high mo-
free of phenol. If either protein or phenol remains lecular weight DNA and if DNA has been degrad-
in the preparation, the respective ratio of absorben- ed completely, the fluorescing band will migrate
cies will fall. However. UV determinations do not with or ahead of the bromophenol blue tracking
enable an estimate of the extent of DNA degrada- dye that is added to each sample. Partially degrad-
tion. ed DNA will be seen as a smear of fluorescing ma-
terial that runs from the high to the low molecular
weight regions of the gel. It is recommended that
Without modification, DNA does not have the standards of RE digested viral DNA be included
ability to fluoresce. Fluorescence measurements on each yield gel as comparative size standards.
can be made only after a suitable dye has been al- For example, RE digestion of lambda phage DNA
lowed to interact with the DNA. Two dyes that by HindIII, yields six DNA fragments that range in
have been used for this purpose are Hoechst 33258 size from 2027 base pairs (bp) up to 23130 bp.
(Brunk et al. 1979) and ethidium bromide. The ex- Yield gels do not enable estimates of protein or
citation and emission maxima for both dyes shift phenol contamination.
when they are bound to DNA. Hoechst 33258 ap- None of the quantitative/qualitative methods
pears to bind preferentially to regions of DNA that described to this point permit estimates of the
are rich in A-T base pairs; whereas ethidium bro- human DNA that is present in a specimen. Since
mide intercalates between the stacked bases DNA other than human can potentially be present
(Watson et al. 1987). Quantification of DNA by flu- in a forensic specimen, these methods can result in
orescence is about l0 times more sensitive than by an overestimate of the quantity of human DNA.
IJV measurement. Fluorescence measurementsonly
permit quantification of DNA, nothing can be Slot,zdot Blot and lluman DNA Probe
learned about the molecular weight of DNA the This method, unlike those previously de-
level of contamination by protein or by phenol. scribed, permits the specific estimation of the
amount of human DNA in a specimen. To carry
Yield Gel Measurements
out the slot/dot blot test, DNA recovered from the
The approximate concentration and molecular specimen is denatured and bound to a nylon mem-
weight of DNA obtained from specimens can be brane. Generally, the membrane is held in an appa-

ratus that enables the application of multiple speci- Ebeling, ll'., Hennrich, N., Klockow, M., Metz, H.,
mens on one membrane. The sample application Orth, H. D. and Lang, H. (1974) Proteinase K
area calohave either the configuration of a dot or a from Tritirachium album limber, Eur. J. Bio-
slot, hence the term, slot blot or dot blot. The chem.47:91-97.
membrane-bound DNA specimens are then hybri-
Gill, P., Lygo, J. E., Fowler, S. J. and llerrett, D. J.
dized with a radioactivelyJabeled DNA probe that
(1987). An evaluation of DNA fingerprinting
is complementary to a highly repetitive sequence
for forensic purposes, Electrophoresis 8:38-44.
found only in human DNA. An example of such a
DNA probe is pl7H8 (Waye et al. 1989) that is Gross-Bellard, M. Oudet, P. and Chamron, P. (1973).
complementary to a highly repetitive sequence lo- Isolation of high-molecular-weight DNA from
cated in human chromosome 17. When this probe mammalian cells, Eur. J. Biochem. 36: 32-38.
is used under low stringency conditions, it will hy- Jeanpierre, M. (1987). A rapid method for the puri-
bridize with closely related sequencesfound in the fication of DNA from blood. Nucleic Acids
DNA of other chromosomes. Because the concen- Res.l5:961l.
tration of the target sequencesis high, many mole- Kanter, E., Baird, M., Shaler, R. and Balazs. I.
cules of probe are bound and an intense autoradio- (1986). Analysis of restriction fragment length
graphic signal can be obtained in just a few hours. polymorphisms in deoxyribonucleic acid
Standards of DNA are placed onto the membrane (DNA) recovered from dried bloodstains, J.
along with the test specimens as quantitative refer- Forensic Sci. 31:403-408.
ences. This technique, although semi-quantitative,
Kirby, K. S. (1957). A new method for the isolation
is extremely sensitive, enabling the detection of
of deoxyribonucleic acids: Evidence on the
human DNA in the pg range. Dot,/slot blot tech-
nature of bonds between deoxyribonucleic acid
niques do not inform the analyst about protein or
and protein. Biochem. J. 66:495-504.
phenol levels in the specimen, nor do they reveal
anything about the intactness of the specimen Lindblom, R. and Holmlund, G. (1955). Rapid
DNA, unless the target DNA is so thoroughly de- DNA purihcation for restriction fragment
graded that the probe will not hybridize. length polymorphism analysis, Gene Anal.
Techn. 5:97-101.
CONCLUSIONS Mann, T. and Lutwak-Mann, C. (1981/. Male Re-
The isolation of DNA from forensic specimens productive Function and Semen, Springer-
is not a difhcult procedure. As with any technique Verlag, Bedin.
however, care must be exercised to insure that the Marashi, F., Stein, G., Stein, J. and Schubert, C.
limited quantities of DNA that are available in (1985). Use of ultrafiltration microconcenrra-
such specimens are not squandered by poor or tors in the concentration and desalting of
sloppy analytical technique. It has been the experi- DNA, Biotechniques May,/June 238-240.
ence of many individuals that improved yields of Marmur, J. (1961). A procedure for the isolation of
DNA are seen as analysts gain experience with, deoxyribonucleic acid from microorganisms, J.
and confidence in, the procedures.
Mol. Biol. 3:208-218.
Newer methods for the recovery of DNA
Miller, S. A., Dykes, D. D. and Polesky, H. F.
from forensic specimens may be on the horizon.
(1988). A simple salting out procedure for ex-
Procedures have been published which show that
tracting DNA from human nucleated cells,
DNA can be recovered effectively while avoiding
the use of hazardous reagents such as phenol. Nucleic Acids Res. 16:1215.
yl/atson, J. D., Hopkins, N. H., Roberts, J. l4t, Steitz,
These procedures utilize high NaCl concentrations
(Miller et al. 1988} or chaotropic agents such as J. A. and Vl/einer,A. M. (1987). Molecular Bi-
guanidinium hydrochloride or urea (Jeanpierre ology of the Gene, 4th ed. The Benjamin,/
1987; Lindblom and Holmlund 1988) to effect the Cummings publishing company, Menlo Park,
removal of proteins from DNA. California.
Waye, J. 5., Presley, L., Budowle, 8., Shutler, G. G.
REFERENCES and Fourney, R. M. (1989). A simple and sensi-
Brunk, C. F., Jones, K. C. and James, T. W. (1979). tive method for quantifying human genomic
Assay for nanogram quantities of DNA in cel- DNA in forensic specimen extraats, Biotechni-
lular homogenates, Anal. Biochem. 92:497-5OO. ques 7:852-855.