DNA fingerprinting

1. BAC DNA preparation
   1. Miniprep with AutoGen 740
      • Innoculate 3-4 ml LB with 20µg/ml chloramphenicol
      • Grow at 37 °C overnight with shaking
      • Isolate BAC DNA with AutoGen 740
      • Resuspend BAC DNA in 50 µl TE pH8.0 or 50 µl ddH₂O
   2. Miniprep with Qiagen kit

2. Check the quality and quantity of miniprep BAC DNA
   1. Digest 5 µl BAC DNA with Hind III
   2. Run in 1% agarose gel
   3. Stain gel with ethidium bromide
   4. Photograph the gel with Stratagene Eagle Eye II Still video system
   5. If DNA was not good enough, repeat the procedure.

3. Fingerprinting Gel
   1. Equipment and materials:
      • Stratagene sequencer - CastAway^TM gel system with gasket lubricant(Cat # 401075)
      • Stratagene castAwaytM precast gels (Cat # 401092): 4.5% precast polyacrylamide, 1xTBE, 7 M urea, with 32 preformed wells
      • High voltage power supplier (> 2000 V)
      • Stratagene castAwaytM gel dryer
      • Molecular dynamics storage phosphor screen (36 X 43 cm)
   2. Marker preparation
      • Digest lambda DNA with Hinf I (or use precut DNA)
      • Label:

              5.0 µl    lambda/Hinf I (10ng/ul)
              4.0 µl    5X AMV-RT buffer (USB, Cat# 70218)
              0.5 µl    32p- dATP
              2.5 µl    3dNTP (-dATP)  (0.33 mM each)
              0.5 µl    AMV-Reverse transcriptase (USB, #700412)
              7.5 µl    ddH2O
              Total 20 µl.
        

        incubate at 42 °C for 15-20 min. Add 20 µl ddH₂O and 80 µl formamide mix (USB sequencing reaction stop solution, Cat.#70704). Aliquot and store at -20 °C. Boil 5 min and transfer to ice before use. Load 1 µl per lane.
   3. Sample preparation
      • Ban I - Msp I double digestion Make mixture: 2.0 µl New England BioLab buffer 2 0.5 µl Ban I (NEB, Cat# 118) 0.5 µl Msp I (NEB, Cat# 116) 0.25 µl RNase A 6.75 µl ddH₂O (for AutoGen miniprep) or 13.75 µl ddH₂O (for Qiagen miniprep) 10 µl BAC DNA (AutoGen miniprep) or 3 µl BAC DNA (Qiagen miniprep) Incubate at 37 °C for 1 hr. Add 50 µl 100% ethanol, vortex, incubate at room temperature for 3 min, and spin at max speed in a desktop centrifuge for 5 min. Air dry the pellets.
      • Label

              Make mixture:
              10.0  µl    5X AMV-RT buffer
               1.0  µl     32p-dATP
               1.0  µl     3dNTP (-dATP)  3.3 mM each
               0.5  µl     AMV-reverse transcriptase
              38.0  µl     ddH2O
        

        Total 50 µl for 1 gel (21 samples). Add 2 µl labelling mixture to each sample, mix very well and keep at 42 °C for 15 -20 min. Add 4 µl of formamide mix (Stratagene sequencing reaction stop solution). Boil 3-5 min aand keep on ice immediately. Load 1 µl(Qiagen prep) - 2 µl (AutoGen prep)
   4. High resolution denaturing gel (Stratagene precast sequencing gel (cat.# 401092)
      • Loading pattern:

        N M S C S M S S S M S S S M S S S M S S S M S S S M S S S M N N
        

        M = Marker S = Sample to be analyzed on the gel C = Internal Control (loaded in a different lane on a different gel) N = any DNA sample (added to prevent lane bending)
      • Gel running Set up gel box at room temperature Running buffer: 1 X TBE Prerunning time: 25 min Run at consistent Watts, 30 Watts per gel Running time: about 85 min; run until the first dye (Bromophenol blue) arrives at low buffer reservoir. (The first dye run ~40 cm.) Run 4-6 gels (80-120 samples don't including internal controls) per day.
      • Fix gel in 10% acetic acid - 10% methanol for 15-20 min. Rinse gel with dH₂O for couple of min to remove urea.
      • Put gel into Stratagene gel dryer to automatically dry the gel (about 40 min).
      • Cover the gel with handi-wrap plastic film , mark the gel number and date carefully and set it into Molecular Dynamics storage phosphor screen cassette and exposure for ~ 40 hrs.

4. Gel imaging
   1. Scan the gel image with Molecular Dynamics PhosphorImager
   2. Pre-process the gel image with ImageQuant (IQ) software
      • Cut the raw image into smaller size and save
      • Convert the above new image from 16-bit to 8-bit tiff format

5. Edit fingerprint image with Image 2.1 or 3.x
   1. For Image 2.1, the image needs to be modified first with XV (John Bradley)
      • Invert the image by 90° so that sample bands run from left to right
      • Set the image size from "a x b" to "2a x (b/2)" or "a x (b/4)"
      • Use color edit function of XV to make a "reverse vedio" of the image (required by image 2.1)
   2. A processed sample image is available for downloading.
   3. If using image 3.x, the 8-bit tiff file from ImageQuant can be used directly. Refer to the Sanger Center for more information and a step by step tutorial.

6. Analyze fingerprint image with FPC 2.5