Anal. Chem. 1002, 64, 2678-2681
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Video Analysis of DNA Sequence Homologies Michael Ludwig’ and Robert J. Hartzman Georgetown University School of Medicine, Department of Pediatrics, Washington, DC 20007,and C. W. “Bill” Young Marrow Donor Recruitment and Research Program, Naval Medical Research Institute, Bethesda, Maryland 20889-5055
A method for the rapld quantitative analyds of dot blot assays k presented. A video camera, an NTSC compatible frame grabber board, and an AT personalcomputer are used to read photographlcexpowresof the assay plate. Image processing and Image analyds techniques are used to calculate the orientation of the dol raster and then to compensate for the effect of variation8 in field illumination on measurements of local contrast. Localcontrast (between dots and background) k an exponential function of the amount of hybridization between blotted DNA and complimentary oligonucleotide probes. The amount of hybridization between blotted DNA and oilgonucleotide probes of known sequence is the criteria wed to estaMirh HLA-DR t h e types. Although the assay described here utilizes a chemliwninescent reaction, this algorithm may be used to read any assay that produces a rectangular raster of dots.
INTRODUCTION Dot blot assays are a standard technique for measuring the amount of biological material bound to a membrane.’ At the C. W. “Bill”Young Marrow Donor Recruitment and Research Program, dot blots are used to evaluate the DNA sequence of human leukocyte antigen (HLA) genes isolated from potential bone marrow donors. The HLA types of donors are entered into a national data base that is used to match the donors with patients requiring bone marrow transplants. A large number of tests are needed to complete the typing of a single individual, hence the requirement for an inexpensive, rapid, accurate, and easy to use method for reading the assay. Dot blot assays are commonly evaluated either visually or by densiometric s ~ a n n i n g .Advancements ~~~ in commercial filtering apparatus have greatly eased sample preparation procedures. However, the tedius, time-consuming, and errorprone tasks of manual analysis and data entry underscore the need for automated analysis. Previous attempts to automate reading have assumed the presence of mainframe computers, required extensive user intervention,334or utilized dedicated systems running proprietary software. In the dot blot assay, the DNA blotted at each dot position was tested against probes of known sequence. Measurements of the degree of hybridization between the probe and blotted DNA were used to evaluate the HLA-DR tissue types of potential bone marrow donors. Our goals in developing this video system were to (1)quickly and accurately read the large numbers of assays produced daily and (2)introduce a measure of numeric consistency that was lacking in visual assessments of the assay. The dot blots were read using a video camera and an image acquisition board to capture the image of a photographic
* To whom correspondence should be addressed.
(1) Kafatos, F. C.; Jones, C. W.; Efstratiadis, A. Nucleic Acids Res. 1979, 7, 1541-1552. (2) Thomas, P. S. h o c . Natl. Acad. Sci. U.S.A. 1980, 77,5201-5205. (3) Williams, R. L.; Cascio, D.; Wight, P. A.; Spindler, S. R. DNA 1985, 4,255-262. (4) Taub, F. E.; DeLeo, J. M.; Thompson, E. B. DNA 1983,2,309-327. 0003-2700/92/0364-2678$03.0010
exposure of an assay plate. The analogue NTSC signal from the video camera was digitized by the frame grabber. Approximately 5 s was required to read and save a single exposure. The results can be observed immediately on the PC screen and may be ported to many commercial databaee programs for further analysis.
EXPERIMENTAL SECTION The dot blot assays were prepared according to standard protocol^.^ Figure 1 illustrates the preparation of a single
(positive) dot. Human DNA was amplified using polymerase chain reaction (PCR) to produce DNA complementary to the sequence of interest. The amount of DNA (absorption coefficient = 0.020 cm2/pg at 260 nm) after PCR was measured with a Spectronic 601 absorption spectrophotometer (Milton Roy, Rochester NY). The PCR was slowlyfiltered through Zeta-Probe membranes (Bio-Rad, Richmond CA). For the calibration experiments, it was assumed that all of the applied DNA adhered to the membrane. This assumption is valid since our maximum target size was 0.09 pg of DNA, while Bio-Rad has loaded targets containing up to 7.5 pg. The surface area of the membranes was sufficiently high to accommodate all of the applied DNA. Samples containing up to 0.0858 pg of PCR-amplified DNA (in 1OX SSPE--1.5 M NaCl, 0.1 M NazHPO4-7Hz0, 10 mM EDTA) were individually blotted to the membrane in a 96-well vacuum filtering apparatus (Bio-Rad). The 96-well microtiter plate sets the 8 X 12 dot raster pattern. The dot positions are labeled A1 (row 1 column 1)to H12 (row 8 column 12). After blotting, the DNA was denatured with0.5 M NaOH, cross-linked to the membrane with a 30-8 1200-& exposure in a UV Stratalinker 1800 (Stratagene,La Jolla, CA),dried, and rewet in 2X SSPE. The membrane was incubated for 60 min in a solution of 0.33% (v/v) 20X SSPE, 0.1% (v/v) Denhardt’s solution (5.0 g of Ficoll Hypaque, 5.0 g of polyvinylpyrrolidone, and 5.0 g of bovine serum albumin in 500 mL of water),0.2g of sarcosine,0.4 mL of 10% SDS, 20.0mg of salmon sperm DNA (Sigma Chemical Corp., St.Louis, MO),and a digoxigenin-labeledsequence-specific 18 base pair oligon~cleotide.~ The probe concentration in the reaction mixture was 100pM. The membranes were then washed (pH 7.5,50-60 “C) to remove nonhybridized and nonspecifically bound oligonucleotide. Detection of digoxigenin-linked oligonucleotide was accomplished as follows. First, nonspecific antibody binding was blocked using a buffered solution of 2 % nonfat dry milk (pH 7.5, Boehringer Mannheim, Indianapolis, IN). The membranes were then incubated with a solution of anti-digoxigenin-Ab-alkaline phosphatase conjugated antibody (BoehringerMannheim). The blots were transferred in a darkroom to a solution of Lumiphos530 (Boehringer Mannheim) and placed in a film cassette. After a final 30-min incubation, the blots were exposed to X-ray film (Kodak X-OMAT scientific imaging film) for timed intervals and developed. Chemiluminescenceresultingfrom the enzymatic cleavage of Lumiphos could then be seen as a dark spot against a clear background. Figure 2 shows the equipment required for the video analysis. The developed films were placed on an illuminated copy stand. A black and white CCD video camera (Cohu, San Diego, CA) ( 5 ) Georgetown University and Naval Medical Research Institute Bone Marrow Typing Standard Operating Procedure. Naval Medical Research Institute: Bethesda, MD, 1991.
0 1992 American Chemical Society
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Flgurr 1. DNA sequence homology test. Human cDNA is linked to a membrane by UV light. A digoxigenln-linked oligonucleotide probe wlli bind to the cDNA if they are homologous. Next, the membrane is washed with aikallnephosphatase bounddlgoxigenln antibody.Flnaily, the membrane Is placed in a solutionof Lumiphos 530. The enzymatic cleavage of Lumlphos produces Ilght.
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Setup for the vIdeo analysis of dot blot assays.
with a Fujinon HF16A CCTV lens was focused on the film. An AT personal computer (Dell Computer Corp., Austin, TX) and a 3Mb P-360 power grabber (Dipix Technologies, Ontario, Canada)was used to capture and analyzethe images. The P-360 is a singlet-slot PC-basedvideodigitizer that uses the TMS320C30 (Texas Instruments, Austin, TX) floating point digital signal processor. The P-360 digitized the NTSC (RS-170 2:l interlace, 60 Hz) video signal. In the calibration experiments, neutraldensity filters were placed between the CCTV lens and the film exposure to simulate changes in room lighting.
RESULTS AND DISCUSSION Program Algorithm. Figure 3 is a flowchart for image acquisition and analysis. The steps requiring user intervention are shown in solid boxes, while the steps automatically carried out are in dotted boxes. The program requires that the first exposure read in each session must be a previously prepared template. This template is simply a developed film image of a microtitre plate with positive spots in diagonally opposite corners (the A1 and H12 positions). These positions were forced positive by using DNA-linked digoxigenin bound to the membrane instead of PCR-amplified DNA solution. There are no other spots on the template. The template is used to establish the geometric parameters required for alignment and to optimize the overall video levels used in reading the ensuing exposures. The program sets the video levels by repeatedly calculating the histogram of the acquired image and adjusting the gain (white clipping level) and offset (black clipping level) of the video amplifier stage
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Flgurr 4. Alignment of a 96 well assay exposure. Pixels are search along R until a second "dot" Is found Identifying the H12 corner. The angle of rotation (a)and the known geometry of the plate are used to calculate the posklons of each well.
of the P-360 until overflow and underflow are eliminated. The video calibration takes about 10 s. The diagonal distance between the two template spots ( R in Figure 4) is measured after the template is read. R is measured by creating a binary image of the template gray image.6 The binary image contains only those pixels below a threshold gray value. Contiguous pixels in the binary image represent the objects that are observed in the image. The size and shape of each group of pixels in the binary image is calculated to determine whether it is one of the reference spots. Once the two reference points are found, R is equal to the distance between them. Since R is a function of the size of the image on the CCD it depends on the distance between the camera and the film exposure. R is measured at the start of each session and must be remeasured if the camera is moved. Other geometrical parameters required for the determination of plate orientation include the length, width, and the number of rows and columns contained in the dot raster. These parameters are set by the geometry of the microtitre plates used and may be chosen a t the beginning of the session. Thus, assays using different raster patterns are easily accommodated. After R has been measured, the dot blot films are successively placed on the copystand. As with the template, each exposure must have dark spots in the A1 and H12 positions. In addition, the A12 and H1 positions must be blank. Figure 4 shows how the dot raster in an image is aligned. A binary image of the plate is created from the captured gray value image. The objects in the binary image are subject to size and shape tests to determine whether they are potentially one of the reference spots. If the size and shape tests are ( 6 ) Gonzales, R . C . ; Wintz, P . Digital Image Processing; Addison-Wesley: Reading, MA, 1987.
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GRAV U A L U E BRIGHTNESS 162 Figure 6. Comparlson of the average gray values of image areas read for a slngle plate under varying llghtlng and amplifier conditions. The slopes of these lines were used to construct Figure 6. Plate brightness values: curve A = 209, B = 149, C = 114, D = 75. passed, the pixels a t a radius R from the center of the spot are searched. If the object is indeed one of the reference spots, there will be one (and only one) position at the radius R that is covered by another object that also passes the size and shape tests. The identify and search process is repeated until both reference spots are located. Once the corner positions are identified, the rest of the exposure orientation is calculated by determining the angle between R and the diagonalof an imaginary‘perfectly aligned” exposure. Figure 4 shows an exposurerandomly placed within the camera field of view (solid rectangle) and the outline of an exposure placed so that its edges are aligned with the camera field of view (dotted rectangle). Using A1 as the origin and the known microtitre plate geometry, the positions of the remaining 94 wells are calculated by a two-dimensional rotation of coordinates through a. At this point the program signals the user that one of the following has occurred: (1)Both reference points have been located. In this case, the exposure will be automatically read. The results appear on the screen and the user is prompted to save them. If saved, the results are written to a file for detailed analysis at a later time. (2) The program is unable to locate two reference points. This may occur if (A) either A1 or H12 is out of the field of view of the CCTV lens and (B) the reference points are of too low contrast to appear in the binary image. The binary image is displayed, so these two cases are easily distinguished. The exposure may be reread after moving it into the field of view or increasing the binary threshold. (3) The reference points are present, but some positions fall out of the field of view of the camera. Alternatively, the program can tell if the exposure has been placed upside down (the reference points are in A12 and H1 giving a very large value for a). In either case, the user is given the opportunity to reposition the exposure. The local contrast a t each spot position is read by sampling a 6 X 6 pixel area at the calculated well position. The background surrounding each spot is sampled by reading up to 864 pixels (a 30 X 30 pixel area excluding the 36 ‘spot” pixels) surrounding the calculated spot position. The standard deviation of both the spot and background pixel
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B R I GHTNESS Local contrast correction factorsas calculated from observed Image brlghtness. Figure 6.
intensities are also calculated. Thus, nonuniform spots and uneven background shadings are easily detected. In addition, dark pixels far from the calculated dot center positionbelonging to dots that are too large or smudged-may be identified and automatically filtered from the background. In practice, the spots produced by the alkaline phosphotase reaction are uniform. Physical disruptions of the membrane during filtering, washing, or pipetting are the primary cause of nonuniform spot density and irregularities of shape. Quantitative Calibration. Measurements of local contrast vary widely with changes in room-lighting conditions. The value of any pixel in an acquired image is also a function of the gain and offset settings of the frame grabber amplifier. Figure 5 is a plot of the average gray values of image areasregions defined by the 36 pixels at calculated dot locations (asdescribed above) and the 864pixels surrounding each dot location that comprise the local backgrounds. The image brightness is defined as the average value of the pixels located outside the boundaries of the microtitre plate. Under normal room illumination the image brightness value was 162. The lines labeled A (verybright lighting, brightness = 209) through D (dark lighting, brightness = 75) are plots of the average gray value of each image area acquired with a brightness level of 162 against the corresponding values of that area obtained under other lighting conditions. In Figure 5, readings from strongly positive dots appear near the origin while readings from the local backgroundsare clustered a t higher grayvalues. Faint dots are distributed along the lines connectingthe origin and the background readings. Figure 6 shows the slopes of a series of such lines as a function of image brightness. Using Figure 6, variations in local contrast caused by fluctuations in room lighting and video settings may be corrected by measuring the image brightness and applying the calculated correction factor to each gray value measurement. The corrected value for local contrast (6) at each spot position is 6 = ( B- P ) / u
(1) where B is the value of the surrounding (background) pixels, P is the value a t the calculated spot position, and u is the correction factor read from Figure 6. Figure 7 shows the quantitative analysis for the hybridization of an oligonucleotide probe. The relative contrast
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
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contribute to the experimental uncertainty to varying degrees. We have therefore found it necessary to prepare a concurrent internal standard membrane (from the nonvariable HLADR region) for each panel of bone marrow donors. This internal standard serves as a measure of the maximum strength of response at each dot position in the assay.
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The A1 and H12 positions are forced positive (saturated black) by using digoxigenin-labeledDNA instead of the usual PCR solution. These positions define the upper limit for local contrast measurements on a given exposure. Increasingthe exposuretime of the X-ray film will decrease the detection limit of hybridized DNA. It was possible to detect 84 & 40 pg of hybridized DNA with a 5-min exposure time. This limit represents the faintest detectable dot produced by a 5-min film exposure time under the conditions used to prepare the assay. On the basis of the averageamount of PCR-amplified DNA applied to a spot position during a sequence homology assay, this corresponds to the detection of about 0.1 9% of the total possible hybridization. The upper detection limit can be further decreased by using less DNA spotted at each position and longer exposure times. This will prevent increases in dot size and smearing due to overexposure. As implied in Figure 7 and eq 3, the detection limit is not a linear function of exposure time. Further, the limit of detection is a function of many experimental variables, each influencing how well hybridization occurs between the probe and the target DNA and how well the blotted DNA adheres to the membrane. The temperature during incubation, the pH of the wash medium, turbulence and shear induced during the wash and filtration steps, the molecular weight of the probe, photographic exposuretime, and the inherent sequence homology between the probe and the blotted DNA each
To demonstrate the utility of the dot blot analyzer, PCRamplified DNA from a panel of 94 individuals was anlayzed. DNA was blotted onto 17 membranes as discussed above. Each membrane was tested for hybridization by a different oligonucleotide probe. The probes were complementary to known sequences of the variable region of HLA-DR. In addition, a membrane was prepared using a probe complimentary to the nonvariable region of the HLA-DR gene. This membrane is used as a measure of the total amount of DNAamplified PCR spotted onto the membrane at each position. After development, the films were read and the data were used to assign donor HLA-DR types. The type assignments made using video data were identical to the types assigned after visual inspection of the assays. The entire panel (over 1700dots) was read, and the data were stored in a commercial database in less than 10 min. Typically, visual inspection of the assays, corroborating the results, and manual data entry take 12-16 h of technician time. As demonstrated above, the video analysis is capable of quantitative analysis. When the assay is read visually, however, quantitative information is not used for type assignments of HLA-DR regions. This is due to the large number of dots that must be evaluated before an assignment is made and the subjectivity of evaluating dot densities by eye. With visual inspection, type assignments are usually made on the basis of the presence of a few dots and must be verified by accomplishedtechnicians who “know the probes”. Cross reactivity (nonspecific probe hybridization) impedes both quantitative and qualitative approaches to type assignments. We are developingmethods for type assignmentsthat combine the quantitative readings obtained by the video analysis with our accumulated experience. These methods are based on a statistical correlation of donor responses with the expected responses from known types.
ACKNOWLEDGMENT Support for this research was provided by the Naval Medical Research Institute Work Unit 63706N.M2022.001. 1110. The work was done as part of the C. W. ”Bill” Young Marrow Donor Recruitment and Research Program. The authors are grateful to Amy Cigan, Caroline Carter, Alicia Reyes, and Pat Mero for preparing the dot blot assays and to Lee Husted for his valuable suggestions and contributions concerning the user interface. Views presented in this paper are those of the authors: no endorsement by the Department of the Navy has been given or should be inferred. RECEIVEDfor review May 18, 1992. Accepted August 24, 1992.
