Current Protocols in Human Genetics
Featured Protocol

Contributed by Paul Meltzer and Michael Bittner
National Institute for Human Genome Research
Bethesda, Maryland

This unit presents protocols for microdissection of human metaphase chromosomes and polymerase chair reaction (PCR) amplification of microdissected chromosome fragments. This procedure and its potential applications (see Background Information) are illustrated in Figure 4.8.1). Of these applications, the generation of fluorescent in situ hybridization (FISH) probes (see Basic Protocol 1) is the one that will stimulate most laboratories to set up microdissection. Using the Basic Protocol 1, it is possible to generate a useful FISH probe for any given chromosomal region starting from banded normal metaphase chromosomes. In addition, and perhaps of greater value, is the application of these methods to the dissection of marker chromosomes which are not readily recognizable by standard banding procedures. A FISH probe prepared by dissecting the region in question can be hybridized back to a normal metaphase revealing the composition of the unknown chromatin. The procedure is rapid, requiring only 3 to 4 days for completion. An additional protocol (see Basic Protocol 2) is included for hybrid selection of region-specific cDNAs that can generate probes for genes transcribed from a microdissected chromosomal region.
 
  BASIC PROTOCOL MICRODISSECTION AND PCR AMPLIFICATION OF BANDED HUMAN CHROMOSOMES

Microdissection is accomplished with glass needles controlled by a micromanipulator attached to a suitably configured microscope. This enables the recovery of band-sized chromosome fragments that are transferred to a collecting drop in an ordinary microcentrifuge tube. Banded metaphase chromosomes from any source are suitable. Larger regions (such as chromosome arms or whole chromosomes) can be dissected by pooling smaller pieces. A multistage PCR reaction primed by degenerate oligonucleotides enables the amplification of these minute quantities of DNA. The resulting PCR product can be converted into a highly robust probe for fluorescent in situ hybridization (FISH).

Materials

Cultured cells

Collection buffer (see recipe)

Human genomic DNA

10 U/µl topoisomerase I

12 U/µl modified T7 DNA polymerase (e.g., Sequenase, version 2) and enzyme dilution buffer

PCR mix (see recipe)

Low-molecular-weight DNA size markers

PCR mix (see recipe) containing biotin nucleotide mix or Spectrum Orange nucleotide mix (APPENDIX 2D)

3 M sodium acetate, pH 5.2

100% ethanol, ice-cold

100 mM Tris.Cl (pH 7.5)/1 mM EDTA

Glass slides or 22 × 60-mm glass coverslips

1.2-mm glass capillary tubes (World Precision Instruments

Pipet puller

Modeling clay

Petri dishes, plastic

0.5-ml thin-walled microcentrifuge tubes, sterile

Gloves sprayed with antistatic spray

Bright-field microscope mounted on a vibration isolation table and equipped with high-resolution micromanipulator (Narashige) and rotating stage

Thermal cycler with heated lid

BioSpin P6 columns (Bio-Rad)

Additional reagents and equipment for mammalian cell culture (APPENDIX 3G), preparation of metaphase spreads from cultured cells (UNIT 4.1), chromosome banding (UNIT 4.2), agarose gel electrophoresis (UNIT 2.7), and ethanol precipitation of DNA (APPENDIX 3C)

NOTE: Exceptional care must be taken in preparing reagents for chromosome microdissection. Each working solution is prepared, divided into single-use aliquots, tested, and used for microdissection only if satisfactory. When preparing solutions, wear gloves and, to the extent it is possible, conduct all manipulations in a laminar flow hood.

Prepare metaphase chromosome spreads

1. Prepare fixed metaphase chromosome spreads from the desired cultured cell source according to standard methods (UNIT 4.1). G-band the chromosomes with trypsin-Giemsa (GTG) prior to microdissection (UNIT 4.2)

   
   To minimize DNA damage, a limited period of fixation (<2 hr) in 3:1 (v/v) methanol/acetic acid prior to making slides is preferred. Metaphases are spread on clean slides or coverslips (22 × 60-mm) and are minimally heat treated (37°C for 2 to 3 days) prior to banding. Exposure of slides to higher temperatures or prolonged storage at ambient temperature reduces the quality of the product from microdissections. A low density of cells is preferred to minimize the film of DNA from lysed interphase cells which may be present. Prepare needles for dissection

2. Using 1.2-mm glass capillary tubes and a pipet puller, pull a sufficient number of glass needles for the planned experiment. Each needle is used once and discarded. For storage, mount needles by their blunt ends on modeling clay strips placed in a plastic petri dish.

   
   CAUTION: The fine microneedles are sharp and require careful handling and storage. Storing the needles mounted on clay strips protects the fragile tips.

   The pipet puller settings will need to be optimized for any given instrument. The ideal needle is substantially coarser than the fine pipets that these instruments can produce for such purposes as intracellular electrodes. If the pipets are too thin and flexible, they will slide over the chromosomes. The tip of the needle should be no smaller than the width of the chromosome or ~0.5 µm.

3. Place the needles in a UV cross-linker and expose for 5 min to deliver ~28 µJ/m² of incident energy. Store irradiated needles in a closed petri dish until they are needed. Dissect chromosomes

4. Prepare four 0.5-ml microcentrifuge tubes containing a 5-µl drop of collection buffer--one for the sample, one for a reagent blank, one for a sham microdissection, and one for a positive control. Add a few picograms of genomic DNA to the positive control tube.

   
   All steps that involve addition of reagents to reaction tubes are carried out in a laminar flow hood to reduce the likelihood of extraneous DNA contamination. It is not necessary to set the microscope up in a hood, although some investigators prefer to do so.

   The collection buffer includes dNTPs and degenerate primer.

5. While wearing gloves sprayed with antistatic spray, mount a dissection needle in the micromanipulator. Place the slide or coverslip containing the metaphase chromosomes on a microscope mounted on a vibration isolation table to stabilize the dissecting needle. Identify a well-isolated example of the target chromosome. Using the rotating stage, position the target region perpendicular to the axis of the dissection needle.

   
   Numerous micromanipulators are commercially available. Most operators prefer the hydraulic design (e.g., Narashige) for chromosome microdissection because of the smooth motion and ease of operation.

   This procedure can be conducted using either an upright or an inverted microscope. If an upright microscope is used, a long-working-distance high-power objective is required such as a Zeiss 50× Epiplan. Using a Zeiss Axioskop, the effective magnification can be doubled with a 2× Optivar (an extra lens in the optical path). For higher-resolution examination of chromosome morphology without oil, a 100× Zeiss Epiplan Neofluar objective can be used. If an inverted microscope is used, the metaphase chromosomes must be prepared on a 22 × 60-mm coverslip.

   Wear gloves during the procedure. Problems encountered with static electricity can be minimized by spraying the gloves with a household antistatic spray.

6. Using the fine controls of the micromanipulator, scrape the needle across the chromosome, staying in the plane of the slide. A fragment will be pushed ahead of the needle but probably will not adhere to it. Collect the scraped fragment by raising the needle, repositioning it above the dissected fragment, and lowering it so that the fragment adheres to the needle when it is raised.

   
   Try to minimize contact between the needle and the slide because extraneous DNA may be inadvertently transferred to the needle.

   Individual operators develop their own "style." With practice, the coarse controls of the micromanipulator can be used to quickly bring the needle into position.

7. Raise the needle with the adherent chromosomal fragment, remove the needle from the micromanipulator, and transfer the chromosome fragment to the collection drop. Discard needle.

   
   The needle tip frequently breaks during the transfer. This does not interfere with the subsequent steps unless the breakage is so high up that the collection fluid is drawn into the broken capillary.

   While learning the procedure, it is useful to replace the needle in the micromanipulator and inspect it to observe the condition of the needle and to be certain that the chromosome fragment is no longer adherent.

8. Place a new needle in the micromanipulator and repeat steps 5, 6, and 7 until the desired number of chromosomes have been dissected.

   
   Although it is possible to store the collection drop at this point, considering the vulnerability of the minute amount of template DNA to traces of nuclease activity, it is best to continue to the PCR step immediately.

   Useful FISH probes can be obtained from even a single dissected chromosome. However, it is not very time consuming to dissect approximately five fragments, and this will increase the chances of success by increasing the amount of DNA available for amplification and compensating for the loss of fragments during the transfer process. However, lengthy dissection sessions should be avoided because efficient PCR amplification depends on rapidly processing the dissected fragments.

9. For a negative control, carry out steps 5, 6, and 7 several times during the dissection, touching the needle only to empty portions of the slide (sham dissection). Transfer these sham dissections to the second blank tube. This will serve as a control for contamination introduced during the dissection procedure.

   
   With sufficient experience, this control should become optional. Relax chromosomal DNA

10. Using a micropipettor, add 1 U (0.1 µl) topoisomerase I to each tube. Incubate in thermal cycler with a heated lid at 37°C.

    
    Topoisomerase I pretreatment appears to render microdissected chromosome fragments more accessible for amplification in subsequent steps by relaxing highly supercoiled metaphase chromosomal DNA.

    If a thermal cycler with a heated lid is unavailable, the collection drop should be overlaid with 20 µl sterilized mineral oil before incubation. Because of the need for multiple additions to the reaction, it is far preferable to avoid the use of an oil overlay.

11. Denature 10 min at 96°C. Preamplify with T7 DNA polymerase

12. Dilute modified T7 DNA polymerase 1:8 with the enzyme dilution buffer provided by the manufacturer to give 1.5 U/µl.

13. Add 0.1 µl diluted T7 DNA polymerase at the annealing step of each cycle to add ~0.15 to 0.2 U each time. Preamplify the DNA using the following program:

    6 cycles:	1 min	30°C
    	3 min	37°C
    	1 min	94°C.

    Although successful results can be obtained without these steps, the topoisomerase I and preamplification steps significantly increase the overall yield of the amplification reaction. The low annealing temperature and high processivity of T7 DNA polymerase increase the efficiency of priming and extension in these critical early extension steps when only minute quantities of template are present. The trade-off in using these additional enzymes is the increased risk of introducing contaminating DNA along with the enzymes. Amplify with Taq DNA polymerase

14. Add 50 µl PCR mix. Amplify the fragment using the following program:

    First cycle:	3 min	95°C	(denaturation)
    35 cycles:	1 min	94°C
    	1 min	56°C
    	2 min	72°C
    Final step:	5 min	72°C	(extension).

    Preparations of Taq DNA polymerase with low DNA content (e.g., AmpliTaq LD, Perkin-Elmer) are available and are preferable for this application.

    Depending on the thermal cycler in use, variations in PCR conditions may produce optimal results. Some investigators utilize a slow ramp from 30°C to 72°C, but this is not necessary if the low-stringency preamplification cycles have been carried out with T7 DNA polymerase.

15. After PCR, analyze 5 µl of the reaction product on a 1% agarose gel containing a low-molecular-weight DNA size marker ladder. Stain the gel with ethidium bromide.

    
    The reaction product should be apparent as a smear in the 200- to 600-bp size range in the experimental and positive control lanes. Both the reagent blanks and the sham dissection control should show little if any DNA in this size range.

    The smear from the microdissection will be very faint. If there is no apparent difference between the reagent blank and the microdissection, this does not necessarily indicate failure, and the experiment should proceed. A strong smear is a greater cause for concern, as this is likely to indicate some degree of contamination. The reagent blank reactions may have a faint smear due to trace contamination or primer dimers/multimers. This is acceptable. The presence of a strong smear in the blank reaction indicates the presence of DNA contamination, most likely in one of the reagents. Label probe for FISH

16. Add 2 µl primary PCR product to a secondary PCR mix containing 48 µl PCR mix in which the standard dNTP mix is replaced with an equal volume of biotin nucleotide mix.

    
    Alternatively, Spectrum Orange nucleotide mix can be substituted for the nucleotides in PCR mix.

17. Amplify and label, using the following thermal cycle program:

    
    15 cycles: 1 min 94°C 1 min 55°C 2 min 72°C Final step: 10 min 72°C (extension).

    For labeling with direct fluorochrome-conjugated nucleotides such as Spectrum Orange (Vysis), better incorporation is obtained with a secondary 20-cycle PCR using standard 4dNTP mix and 2 µl of the primary PCR product as template. 2 µl of this secondary PCR product is then used for a labeling reaction for 15 cycles using Spectrum Orange nucleotide mix.

    Because the labeling amplification contains a much higher template quantity than the primary PCR, ordinary commercial PCR buffer and routine grades of Taq DNA polymerase can be used. Other substituted nucleotides such as digoxigenin or other fluorochrome-conjugated nucleotides can also be used. The optimum concentration of each substituted nucleotide in the PCR mix must be determined empirically, because they are not utilized by the polymerase with equal efficiency. The choice of nucleotides will depend on the availability of appropriate filter sets and the preference of the laboratory. Direct fluorochrome-conjugated nucleotides offer an advantage in terms of low background and simplicity (because they can be examined immediately after washing). However, they are more costly and do not all incorporate well into DNA. Spectrum Orange dUTP does incorporate well and generates a bright signal that can be visualized with a rhodamine filter set. Recover labeled probe

18. Remove unincorporated nucleotides from the product by centrifugal gel filtration on a BioSpin P6 column following the manufacturer's instructions  ( also see APPENDIX 3E).

19. Precipitate the probe by adding 5 µl of 3 M sodium acetate, pH 5.2, and 150 µl ice-cold 100% ethanol (APPENDIX 3C). Microcentrifuge 5 min at 10,000 × g, 20°C.

20. Aspirate supernatant and dry residual DNA pellet for 5 min in a Speedvac evaporator. Resuspend labeled probe in 25 µl of 10 mM Tris·Cl (pH 7.5)/1 mM EDTA. Store at -20°C or below.

    
    Approximately 100 ng of this probe is used in a standard FISH protocol (UNIT 4.3).

    Even if the primary goal of the experiment is to construct a microclone library, preparing a FISH probe is the fastest way to test the quality of the PCR product. If a detectable but weak FISH signal is obtained, this is a satisfactory starting point for fine tuning the protocol to optimize microdissection and PCR (see Table 4.8.1 for troubleshooting information). If no FISH signal is obtained from a PCR product that appears to have the correct size distribution on an agarose gel, there are serious contamination problems that need to be identified and corrected.

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    Figure 4.8.1

    Outline of the microdissection procedure. Chromosome fragments are first dissected and transferred to a collection drop. After topoisomerase I treatment, the DNA is preamplified with T7 DNA polymerase. The amplification is then continued with  Taq DNA polymerase. An aliquot of that reaction (MDN, microdissection) and a blank (to control for DNA contamination) are examined by agarose gel electrophoresis. If the results show a satisfactory smear of products in the 200- to 600-bp range with a negative blank, then an aliquot can be amplified for fluorescent in situ hybridization (FISH), radioactive labeling, or cloning.

    

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    Table 4.8.1 Troubleshooting Guide for Microdissection and PCR

    Problem	Solution
    No PCR product in any reaction	Try new batches of enzymes; replace agents one by one.
    Strong PCR product in blank	Try new batches of enzymes; replace reagents one by one.
    Weak FISH signal	Increase number of fragments dissected; prepare fresh metaphases; obtain new batch of primer.
    Nonspecific FISH signal on centromeres or chromosome arms	Make sure PCR blank is negative; prepare fresh methaphases at lower density.

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