2. Genomic DNA amplification

The exon scanning techniques described in this chapter are all based on PCR. Genomic DNA is extracted from peripheral blood samples by conventional means (11,12). The test DNA is then amplified by PCR (refs. 1,13 and Protocol 1) and the resulting amplification product used in the chosen mutation screening technique. The quality of the amplified DNA can determine the success of the method and the clarity of the results, especially for SSCP. The selection of oligonucleotide primers for PCR is very important. Primer pairs with a similar dissociation temperature (Td) will give a cleaner product since the annealing temperatures will be similar for both. As a rough guide, primers with a Td of 60oC will anneal to their template at approximately 55oC. Trial amplification with a control DNA is recommended to fine tune the reaction conditions. Non-specific background bands can often be eliminated by increasing the annealing temperature by 1 - 2oC. When very little genomic DNA is available and needs to be conserved (e.g. the patient is deceased), secondary PCR reactions can be seeded from the primary amplification. This enhances the yield of PCR product and leaves the genomic DNA in reserve. The same oligonucleotides can be used for both primary and secondary PCR reactions. Any mutation detected must be confirmed by going back to the genomic DNA sample and sequencing a freshly-amplified PCR product. In this way possible infidelities introduced by the Taq polymerase enzyme may be excluded.

Protocol 1. PCR amplification of genomic DNA

Equipment and reagents

Thermal cycler

Positive displacement pipettes, capillaries and pistons (Microman, Anachem)a.

0.5ml sterile microcentrifuge tubes.

100mM dNTPs in sterile distilled water (Pharmacia #27-2035-01).

Taq DNA polymerase 5 units/æl (Promega #M 1862)

1 x PCR buffer : 10 x buffer is supplied with the enzyme and should be diluted with sterile distilled water. Add 20æl each of 100mM dATP, dGTP, dCTP, dTTP for each 10 ml of 1 x buffer. The buffer will now contain 50mM KCl, 10mM Tris-HCl pH 8.8, 1.5mM MgCl2, 0.1% Triton X-100 and 200æmol of each dNTPb. Aliquot in 1ml amounts and store at -20oC.

Oligonucleotide primers 200-300ng/æl.

Mineral oil (Sigma molecular biology grade M5904).

Genomic DNA 400ng/æl.

10 x TBE buffer, pH 8.0 (0.9M Tris-HCl, 0.9M boric acid, 20mM EDTA)

Method

Thaw all the reagents and keep on ice.

1. Prepare sufficient 1 x PCR master mix for the required number of reactions (100æl per reaction). To do this, add 10æl of each oligonucleotide primer and 4æl Taq DNA polymerase per ml of 1 x PCR buffer. Mix well by inversion.
2. Aliquot 100æl of the 1 x PCR master mix into each 0.5ml sterile microcentrifuge tube.
3. Add 1æl of genomic DNA and mix by tapping gently.
4. Overlay each reaction with 50æl of mineral oil. The PCR reaction mixture will now contain 400 ng genomic DNA 200-300 ng of each oligonucleotide primer 2 units Taq DNA polymerase
5. Include a negative (no DNA) control with each batch of amplifications.
6. Programme the thermal cycler. The following conditions are a guide: Final extension 72oC 10 mins Soak 10oC

   Step	Temp	Time
   Denature	94oC	5 mins
   Denature	94oC	35 secs
   Anneal	47-59oC	1 min 30 cycles
   Extend	72oC	1 minc

   
   
   The annealing temperature will depend on the oligonucleotide primer sequences.
   
   
7. Check the amplification by running 10æl of the PCR product on a 1-2% agarose gel in 1 x TBE. aFilter tips are now also available for most pipettes to avoid aerosol contamination. bMgCl2 is sometimes supplied separately to the 10 x PCR buffer and should be added at this stage. cAn additional 3 sec/cycle extension time is often recommended for long amplification products but this is not always essential.
   
   

3. Single Strand Conformation Polymorphism (SSCP)

The mobility of single-stranded nucleic acid molecules, electrophoresed under non-denaturing conditions is determined by both their fragment length and their secondary structure which is sequence dependent (6,14). A fragment may adopt several conformations for any given set of electrophoretic conditions and these are visualised as separate bands in the gel. Some or all of these bands may show a shift in a mutated sequence, a single base change being sufficient to alter secondary structure and hence mobility.

Screening genomic DNA for unknown mutations is now possible, provided that the gene under scrutiny has already been at least partially sequenced to enable oligonucleotide primers to be designed. Selected regions of very large genes, for example the factor VIII gene, consisting of 186Kb of genomic DNA and 26 exons, can now readily be screened. However, this method is equally applicable to any gene however large or small. Even when only cDNA sequence is available, it is possible in some cases to screen small genes at the genomic level by designing primers within the known coding sequence and amplify across introns. Fragments can then be directly sequenced, the only limit being intron size and the limits of the PCR reaction.

3.1 Standard SSCP

A wide variety of electrophoresis and gel conditions for SSCP have been cited in the literature (15); polyacrylamide concentrations varying from 4-6%, differing gel cross- linking ratios, the presence or absence of glycerol in the gel, and electrophoresis at either room temperature or 4oC. The addition of glycerol allows gels to be run at room temperature but with a considerable increase in electrophoresis time. Cooling to 4oC can produce erratic results when cooling fans are used, if a cold room is available carrying out electrophoresis at 4oC is the best option.

The most important criteria for successful SSCP are as follows:

1. DNA fragments should be less than 300bp, since mobility shifts may not be detected in larger fragments.
2. Polyacrylamide gels should be prepared with a low cross-linking ratio such as 39:1 acrylamide to bisacrylamide. Protocol 2 describes a suitable procedure.
3. A stable temperature environment must be maintained during the electrophoretic run, preferably in a cold room at 4oC.
4. Use 33P -dATP which will give crisper bands during autoradiography than 32P - dATP (9). The latter, gives more diffuse bands because of its higher energy of emission. These diffuse bands can obscure minor shifts of mobility of fragments eg. 1-2mm.
5. Gels should be run for a sufficient length of time to enhance the possible shifts. It is also highly advisable to run samples for two different lengths of time (e.g. 3h and 4h) to minimise the risk of missing band shifts. The exact times chosen for electrophoresis depend on fragment length.
6. Always run a double-stranded DNA control. This will enable identification of single- and double-stranded fragments, especially when samples have been digested with restriction enzymes and several fragments are visualised. Band shifts can frequently be seen with double-stranded fragments and sometimes are only seen with the double-stranded fragments.

Protocol 2 Preparation of a polyacrylamide gel for SSCP

Equipment and Reagentsa WARNING: Acrylamide and bisacrylamide are known neurotoxins and potential mutagens and teratogens in man. Always wear disposable plastic gloves, a dust mask and safety glasses during handling of these reagents in powder form and plastic disposable gloves at least when handling their solutions.

40% acrylamide-bisacrylamide (39:1) stock solution.b Prepare by mixing 35.1g acrylamide (Electran, Grade 1, BDH 44313 5B), 0.9g N,N'- methylenebisacrylamide (Life Technologies 540-5516UB) and 90ml distilled water. Dissolve at 65oC for 10 min

4.5% acrylamide stock solution. Prepare by mixing 90ml 40% acrylamide- bisacrylamide (39:1), 80ml 10xTBE buffer (see Protocol 1), and 630ml distilled water. Store at 4oC

10% ammonium persulphate (Sigma A9164)

TEMED (Sigma T7024)

Polyacrylamide gel mould (or 5mm thick float glass sequencing plates) using 0.4mm spacers and 0.4mm comb

Method

1. Filter the 4.5% acrylamide solution through a 0.45æ filter (Sartorius) just before use. Degas the solution under vacuumc.
2. Mix: 4.5% acrylamide solution 80ml 10% ammonium persulphate 460æl TEMED 60æl
3. Pour the gel immediately and allow it to polymerise. aIt is important to use only the highest quality reagents. bThe 39:1 ratio of acrylamide to bisacrylamide is very important since the detection of mobility shifts will be decreased if lower ratios are used. cDegassing prevents the formation of bubbles in the polymerised gel.

Protocol 3

describes a procedure for standard SSCP which satisfies all of these criteria. The conditions used in this protocol have produced a greater than 95% success rate in detecting mutated sequences in our laboratory. Results obtained from the standard SSCP protocol described here are illustrated in Figs. 1 and 2.

Protocol 3. Standard SSCP

Equipment and reagents

High voltage power supply (e.g. Multidrive XL, Pharmacia).

Vertical gel electrophoresis apparatus (e.g. Model S2, Life Technologies 580- 1165SC or similar).

Heated driblock.

à33P -dATP (1000-3000 Ci/mmol). (Dupont NEN #NEG-312H).

Restriction enzymes as required.

Sample running buffer. For single stranded DNA samples, this is prepared by mixing (per 1ml buffer), 800æl formamide, 100æl of 1% Bromophenol blue, 100æl of 1% Xylene Cyanol, 2æl 0.5M EDTA, 1æl 10M NaOH.a Double stranded DNA samples require the same sample buffer but without NaOH.

4.5% polyacrylamide gel (poured using 0.4mm spacers and a 0.4mm comb; see Protocol 2).

Agarose gel (1-2%).

Method

1. 1. Amplify the test DNA fragment (Protocol 1) in the presence of a radiolabelled nucleotide (e.g. 3æCi à33P -dATP) per 100æl reaction). This will label the PCR product during amplification. Some methods involve end-labelling the PCR primers by treating with polynucleotide kinase using 32P -ATP and so generate fragments which are only end-labelled. This is more laborious than the method described here and cannot be used where larger amplification products need to be digested since internal digestion products are then not labelled.
2. Confirm the amplification by running 10æl of the PCR product on a 1-2% agarose gel.
3. Digest fragments larger than 300bp with suitable restriction enzymes.
4. Take 5-10æl of the labelled PCR product and make up to 25æl with sample running buffer.
5. For single-stranded DNA only, denature the samples at 90-95oC for 5 mina and transfer immediately to ice.
6. After 5 min on ice, load 3-8æl of each sample onto the 4.5% polyacrylamide gel. (Vary the sample volume according to the efficiency of amplification, whether the samples have been restricted or not, and the specific radioactivity of the label used).
7. Electrophorese the gel in 1 x TBE at 4oC (preferably in a cold room) and at 40W constant power for 2-6h depending on the DNA fragment sizes. In a 4.5% polyacrylamide gel, Xylene cyanole migrates at a position expected for single- stranded DNA fragments of 210 nt long.
8. After 1 h, load a second batch of the same samples as in step 6 above. By running for two different periods, the risk of missing shifted bands is reduced.
9. A lane of double-stranded DNAa should be run with each batch of samples to enable distinction between single-stranded and the double-stranded bands.
10. Transfer the gel to Whatmann 3MM paper, cover with cling film and dry under vacuum at 80oC for 30-45 min.
11. Expose the gel to X-ray film at room temperature without a screen. Develop the autoradiograph.
    
    a Double-stranded DNA should be prepared in the same way as single-stranded DNA except that it is made up in sample running buffer without NaOH and the sample is not preheated before applying to the gel.

3.2 Consecutive

SSCP

As stated earlier, large PCR fragments (e.g. 1kb in length) have to be digested in order to detect a mobility shift by standard SSCP. However, the localisation of a mobility shift can then prove to be difficult because multiple bands are visualised on the gel. Fortunately, the accurate assignment of a mobility shift can be achieved in such situations by applying consecutive SSCP (16).
The DNA fragment to be screened is amplified in the presence of an à-33P dNTP and using a biotinylated antisense primer (Protocol 4). The PCR product can then be bound by its biotinylated 3' end to streptavidin coated- magnetic beads. A magnetic separator is used to remove the biotinylated product from the unincorporated primers, dNTP's and radiolabelled nucleotides ready for restriction enzyme digestion. Careful selection of restriction enzymes allows sequential digestion of the biotinylated product and the individual collection of the restricted DNA fragments. The restriction enzymes are chosen so as to generate suitable DNA fragment sizes (100-300bp) in a consecutive manner (see Figure 3). SSCP is then performed with each separate DNA fragment loaded individually onto the gel. In this way, previously observed shifts can be accurately assigned to a precise DNA fragment for subsequent direct sequencing. This adaptation of SSCP is therefore the approach of choice for rapid mutation screening of larger segments of DNA.

Protocol 4. Consecutive SSCP

Equipment and reagents

Streptavidin-coated magnetic beads (e.g. Dynabeads M-280 Streptavidin Dynal #112,05)

Magnetic particle concentrator (e.g. Dynal MPC-E #120.04)

PCR primers. The 3' antisense primer must be biotinylated on its 5' end

Radiolabelled nucleotide e.g. à-33P -dATP (Dupont NEN)

Selected restriction enzymes. Choose these so as to generate suitable DNA fragment sizes (100-300bp) in a consecutive manner (see Figure 3).

Sample running buffer (see Protocol 3)

2 x washing and binding buffer (WBB) (10mM Tris-HCl pH 7.6, 1mM EDTA, 2M NaCl)

4.5% non-denaturing polyacrylamide gel (see Protocol 2)

Agarose gel (1-2%)

1 x TBE buffer (see Protocol 1)

Method

1. Amplify the DNA (see Protocol 3) using 100-150ng of the biotinylated primer per 100æl reaction and 200ng of the unbiotinylated primer. Remember to add 3æCi of à33P -dATP to the 100æl reaction mix.
2. Run 10æl on a 1-2% agarose gel to confirm amplification.
3. Wash 40-50æl of magnetic beads twice with 1 x WBB using the magnetic separator to remove the beads from the supernatant after each wash.
4. Resuspend the magnetic beads in 50æl of 2 x WBB.
5. Add 90æl of PCR product to the beads and mix gently by pipetting. Do not vortex. Less PCR product can be used if the amplification is very efficient.
6. Incubate at 37oC for 10 min. This will bind the biotinylated PCR product to the magnetic beads. Incubation at room temperature is possible, but can be less efficient as the ambient temperature is variable.
7. Use the magnetic separator to remove the WBB buffer from the magnetic beads. Pipette off the buffer completely.
8. Add 17æl sterile distilled water to the beads. Mix gently by pipetting.
9. Add 2æl of the appropriate 10 x restriction buffer and 1æl of restriction enzyme. Mix by gentle pipetting.
10. Incubate at 37oC for 1 h.
11. Separate the magnetic beads from the supernatant using the magnetic separator. Transfer the supernatant to a clean 1.5ml microcentrifuge tube and keep on ice. This contains fragment 1.
12. Wash the beads once with sterile distilled water and resuspend in 17æl of sterile distilled water ready for the next digestion.
13. Repeat steps 9-12 as required until all the DNA fragments have been restricted leaving the final fragment still attached to the beadsa.
14. Take 5-10æl of each restricted fragment including the final segment bound to the beads and make each one up to 20æl in sample running buffer.
15. Denature the DNA samples at 90-95oC for 5 min. This will also cleave the final DNA fragment from the beads.
16. Immediately transfer to ice for 5 min.
17. Load 3-8æl of each sample (single-stranded and double-stranded DNA) onto the gel.
18. Run the gel in 1 x TBE at 4oC (preferably in a cold room) at 40W, constant power for 2-6 h depending on fragment length.
19. Continue as for Protocol 3, steps 10-11.
20. Examination of the developed autoradiograph will reveal the shift in one specific restricted fragment. This fragment can then be directly sequenced (see Protocol 6).
    
    a2æl of each completed digest can be run on an agarose gel to confirm that the reactions have gone to completion. This information can be useful during interpretation of the consecutive SSCP gels.
    
    
    
    

3.3 PCR-SSCP multiplexed analysis; application to the factor VIII gene

The aim of multiplexed analysis of very large genes like the factor VIII gene is to speed up SSCP analysis by amplifying several gene fragments simultaneously by the PCR method. All essential coding regions of the factor VIII gene (exons 1-13, 15-26) can be amplified as individual exons including intron/exon splice junctions. PCR fragment sizes range from 143 bp (exon 20) to 352 bp (exon 8). Exon flanking sequences range from 27- 88 bp. Multiplexed PCR amplification was carried out in eight groups: three groups of 4 exons, three groups of 3 exons and two groups of 2 exons were multiplexed at an annealing temperature of 50oC (Table 1). The exons within each group were separated by appropriate size differences and by large genomic map distances (the shortest distance between exon fragments being 5.63 Kb) to avoid artifacts resulting from intronic sequence amplification.

The advantages of the method are fourfold;

speed of analysis

economy of reagents and sample

simplicity of method

sensitivity of detection

The whole of the essential coding region of the factor VIII gene is PCR amplified in only 8 tubes. All primer pairs anneal at the same temperature (50oC). Thus a single patient DNA sample and a control (16 tubes) could have the essential coding region of the factor VIII gene amplified by the PCR-multiplexing system in about 4-5h. All fragments are suitably small enough for SSCP gel analysis without prior restriction enzyme digestion. Thus, the preparation, running and drying of the gel, and the exposure of the dry gel to X-ray film determine the time taken before results are available. With appropriate forethought, results could be available within three days.

Multiplexed amplification is more economical on sample and reagents than individual PCRs performed for each exon; less Taq polymerase enzyme, radioactive isotope (making the procedure safer), nucleotides and buffer are used, and considerably less patient genomic DNA sample is required. The multiplexed PCR-SSCP analysis described here (Protocol 5) is essentially a two step procedure, each strand of the target DNA sequence is labelled as it is amplified in the thermal cycler and then transferred directly to electrophoresis on easily prepared gels. Since both strands of multiplexed PCR fragments are labelled, SSCP analysis may reveal a shift in either of the amplified complementary DNA strands after denaturation or in the double-stranded DNA product (Figure 4).

Protocol 5. Multiplexed PCR

Equipment and reagents

Genomic DNA

Oligonucleotide primers for PCR

Taq DNA polymerase (e.g. Promega)

à-33P -dATP (1000-3000 Ci/mmol; 10 Ci/ml) (NEN Radiochemicals)

1 X PCR buffer (Promega) containing 200æM each of dATP, dCTP, dGTP, dTTP (Pharmacia)

Mineral oil (Molecular biology grade: Sigma)

Thermal cycler (Perkin-Elmer Cetus)

2% agarose gel

Method

1. Use the oligonucleotide primers directly after uncoupling and deprotection. Mix them in groups (as in Table 1 for the factor VIII gene), dry down and reconstitute such that 1æl contains 200ng of each primer.
2. For PCR, add 400-800ng genomic DNA, 200ng of each primer, 1.25 units Taq DNA polymerase/primer pair and 0.25æl of à-33P -dATP/primer pair to 100æl of PCR buffer containing 200æM of dNTPs.
3. Overlay the reaction mixture with mineral oil.
4. Carry out PCR. The PCR conditions for a Perkin-Elmer Cetus thermal cycler are: Denature at 94oC for 5 min. Then perform 30 cycles of PCR as follows: denaturation 94o for 30 sec annealing 50oC for 1 min extension 72oC for 1 min with 3 sec additional elongation per cycle. The final extension is 72oC for 10 min.
5. Check 10æl of the PCR product on a 2% agarose gel to confirm amplification.
6. Continue as in Protocol 3, steps 4-11.

4. Direct sequencing of biotinylated PCR products using Streptavidin coated magnetic beads in a solid support system

Once a suspected mutation is detected using the exon scanning techniques described in this chapter, confirmation is required by DNA sequencing. The DNA fragments could be cloned and sequenced using traditional methods. Although easily achievable using T-vectors (17) or blunt-end ligation, the cloning of PCR products is time- consuming. Furthermore, in order to exclude artifacts introduced by the infidelity of the Taq polymerase during amplification, several clones must be sequenced from each DNA sample being analysed.
This tedious approach can be avoided by direct sequencing of the PCR products. The DNA template must be clean and the excess primers and excess dNTP's must be removed. Various column, gel purification, and electroelution methods have been described but again these are time-consuming and often good readable sequence is difficult to achieve.
A very elegant approach which avoids these problems utilises Streptavidin- coated magnetic beads (18,19) in a solid phase system which is both rapid and reliable. Efficient concentration of the PCR product while simultaneously removing excess primers and nucleotides results in excellent readable sequence.
Amplified DNA template prepared using this solid phase system can be the initial step to incorporate into Taq cycle sequencing in a thermal cycler using Taq DNA polymerase (20), or T7 DNA sequencing (21) in a conventional dideoxy chain termination protocol (22). Both variations are easily adapted for automated DNA sequencing simply by substituting four fluorescently-labelled nucleotides in place of a radiolabelled nucleotide (usually à35S -dATP).
In Protocol 6 we describe the direct sequencing of biotinylated PCR products by preparing single-stranded DNA templates and directly sequencing them using T7 DNA polymerase. Both DNA strands can be sequenced in this way. A schematic representation is shown in Figure 5.

Protocol 6. Preparation of single stranded DNA template and direct sequencing using Streptavidin-coated magnetic beads and biotinylated PCR products.

Equipment and reagents

Genomic DNA

PCR primers with the 3' antisense primer biotinylated at its 5' end.

Streptavidin-coated magnetic beads (e.g. Dynabeads M280- Streptavidin, Dynal 112.05).

Magnetic particle concentrator (MPC) (e.g. Dynal MPC-E, 120.04).

2 x washing and binding buffer (WBB) (see Protocol 4).

0.1M NaOH.

Internal primers for sequencing (20ng/æl).

T7 sequencing kit (e.g. Pharmacia 27-1682-01) and sequencing polyacrylamide gel. (Taq cycle sequencing can also be used).

Agarose gel (1-2%).

Amplification

1. Amplify the genomic DNA using only 100-150ng of the biotinylated 3' primer and 200ng of the 5' primer per 100æl reaction. There should be little or no primer left after amplification.
2. Run 10æl on a 1-2% agarose gel to check amplification. Binding of PCR product to the Streptavidin-coated magnetic beads
3. Pipette 50æl of the beads into a 1.5ml sterile microcentrifuge tube and remove the supernatant.
4. Resuspend the beads in 50æl of 1 x WBB. Mix gently. Discard the supernatant.
5. Repeat the wash using 1 x WBB. Discard the supernatant.
6. Resuspend the beads in 50æl of 2 x WBB. Vary the volume according to the quantity of PCR product. The final concentration of NaCl should be 1-2M for efficient binding of PCR product to the beads.
7. Add an equal volume of PCR product i.e. 50æl. Mix gently. Incubate at 37oC for 10 min. Cool the tubes to room temperature.
8. Discard the supernatant and resuspend the beads in 50æl of 1 x WBB. Mix gently. The PCR/magnetic bead complex can be stored for up to 1 week at 4oC prior to sequencing.

Preparation of single-stranded DNA template

1. Discard the 1 x WBB.
2. Add 50æl of 0.1M NaOH and incubate at room temperature for 10 min.
3. Remove the supernatant and keep this. This contains the DNA sense strand for subsequent sequencing (see below).
4. Wash the beads with 0.1M NaOH. Discard the supernatant.
5. The Streptavidin coated magnetic beads are now bound to the biotinylated antisense DNA strand. Wash the beads once in 50æl of 1 x WBB and then once in 50æl H2O.
6. Resuspend the beads in 10æl H2O ready for sequencing.

DNA sequencing.

1. Using an internal primer, sequence the antisense single-stranded DNA template using the T7 sequencing kit following the manufacturer's instructions for single-stranded DNA. This will generate sense strand sequence.
2. Just prior to loading the polyacrylamide sequencing gel, heat the sequencing reactions at 72oC for 2-5 min. Load 3æl per lane. If required, the magnetic beads can be kept to one side of the tube using the MPC to facilitate easier gel loading.
3. The sense strand template (see step 3 in the previous section) can be sequenced after neutralising with an equal volume of 0.1M HCl and subsequent ethanol precipitation. Redissolve the DNA in 10æl H20 and continue with the sequencing reactions.

(a)Use the MPC to isolate the beads from the supernatants between steps. (b)Only mix by gentle pipetting. (c)Agitate the beads during incubation steps to ensure contact of the beads with the reagents.

5. Chemical cleavage detection of mismatched base pairs

This technique is based on the ability of hydroxylamine and osmium tetroxide (OsO4) to modify single base mismatches (5,23). This is used to identify point mutations at the genomic level by amplifying the DNA fragment of interest, creating a heteroduplex with the radiolabelled wild-type DNA and subjecting the heteroduplex to chemical modification and cleavage.
Hydroxylamine at pH 6.0 modifies the C5=C6 double bond in cytosine and OsO4 oxidises the C5=C6 double bond in a thymine specific reaction. At the point of mismatch in the heteroduplex, there is effectively single-stranded DNA which is readily recognised and modified. Piperidine is subsequently used to cleave at the modified points of mismatch. Hydroxylamine modifies C-C, C-T and C-A mismatches. OsO4 modifies T-G, T-C and T-T mismatches. However by forming heteroduplexes between the mutant and wild-type sequences, all 12 possible point mutations and deletions give rise to at least one detectable mismatch or loop out..
After chemical modification and cleavage, the DNA heteroduplexes are run on a urea/polyacrylamide gel and mutations are identified by the presence of cleaved DNA fragments. Examples are shown in Figure 6 where 3 mutations have been identified in Exon 11 of the factor VIII gene. The size of the cleaved product gives positional information regarding the mutation which is confirmed by sequencing..
Protocol 7 describes the preparation of 6% urea/acrylamide gels for chemical cleavage analysis and Protocol 8 describes the cleavage detection procedure itself. .

Protocol 7 Preparation of 6% urea/acrylamide gels for chemical cleavage analysis

Equipment and Reagents

30% stock acrylamide solution. Prepare by mixing 28.5gm acrylamide, 1.5gm N,N'-methylene bisacrylamide and making up to 100ml with sterile distilled water. Store at 4oC.

Urea buffer. Prepare by mixing 210gm urea and 50ml 10 x TBE buffer (see Protocol 1). Make up to a total volume of 400ml with sterile distilled water. Dissolve by heating at 50oC and filter through Whatman No. 1 filter paper. Store at 4oC.

TEMED

10% ammonium persulphate

Polyacrylamide gel mould with gradient spacers (0.4-1.2mm thick) and a 0.4mm comb.

Method

1. Prepare the 6% urea/acrylamide gel mixturea by mixing: Urea buffer 96ml 30% acrylamide 24ml TEMED 54æl 10% ammonium persulphate 960æl
2. Pour the gel immediately and allow it to polymerise. a The quantity prepared here is for a gel of dimensions 394 x 300mm.

Protocol 8. Chemical cleavage detection of mismatched base pairs. Equipment and reagents

Precipitation solution (0.3M sodium acetate pH 5.2, 0.1mM EDTA)

Ethanol (100% and 70%)

32P -ATP (4500 Ci/mmol) (ICN Radiochemicals)

T4 polynucleotide kinase (Pharmacia #27-0736-02)

Kinase buffer (500mM imidazole-HCl, 100mM MgCl2, 1mM EDTA, 50mM DTT, 1mM spermidine, 3mM ADP)

Nick columns (Pharmacia #17-0855-02 or similar Sephadex G50 columns)

5 x HET buffer (1.5M NaCl, 0.5M Tris-HCl pH 8.0)

Hydroxylamine hydrochloride (Aldrich #25,558-0)

Diethylamine

1M Tris-HCl pH 8.0

500mM EDTA

Pyridine (Aldrich #18,452-7)

4% OsO4 solution (Aldrich #25175-5)

tRNA (10mg/ml), from yeast (Sigma #RO128)

1M Piperidine (Sigma P5881, supplied as 10M, dilute in sterile distilled water to 1M just before use)

1Kb DNA size marker ladder (GIBCO-BRL) and/or íX174 HaeIII fragments

1 x TBE buffer (see Protocol 1)

1% agarose gel

6% urea/acrylamide gel (see Protocol 7)

Stop solution (80% formamide, 0.1% Bromophenol Blue, 1mM EDTA, 10mM NaOH)

Methanol/dry ice bath

Speed Vac (Life Sciences International)

High voltage power supply

Screw capped tubes (1.5ml, Sarstedt)

Amplification

1. Design the oligonucleotide PCR primers within the intron sequences to enable analysis of whole exons and splice junctions. Allow 20-100bp between the primer and the 5'/3' end of each exon; otherwise the cleavage products may be too small to be detected.
2. Amplify the test DNA's (see Protocol 1) and include two normal DNA samples, one to be used as a labelled probe, the other as a negative control. Purification of the normal DNA fragment for use as the probe.
3. Run the total PCR product on a 1% agarose gel at a constant 30V for 1-2 h, in 1 x TBE containing 1æg/ml ethidium bromide.
4. View the DNA fragment using a UV transilluminator and cut out the gel slice containing the DNA fragment. Place the gel slice into a 1.5ml sterile microcentrifuge tube.
5. Place the tube in a dry ice/methanol bath for 30 min.
6. Centrifuge at 14,000 rpm for 30 min.
7. Transfer the supernatant to a clean 1.5ml sterile microcentrifuge tube and concentrate in a Speed Vac until the volume is reduced to 100æl.
8. Add the following:
   
   200æl precipitation solution
   750æl 100% ethanol Place in a dry ice/methanol bath for 30 min
9. Centrifuge at 14,000 rpm for 30min. Wash the pellet in 1ml of 70% ethanol.
10. Dry the pellet in the Speed Vac and store at -20oC until required.

Labelling the probe.

1. Dissolve the DNA pellet in 10.5æl sterile distilled water.
2. Add the following:
3. 10æl 32P -ATP (equivalent to 100æCi)
4. 2æl T4 polynucleotide kinase
5. 2.5æl kinase buffer Mix and incubate at 37oC - for 35 min.
6. Remove the unincorporated radioactivity by passing the reaction through a Nick column.
7. Dry the probe in a Speed Vac and then redissolve in 75æl sterile distilled water. Count 1æl - in a liquid scintillation counter.
8. Use 3-5 x 105 dpm/sample for the heteroduplex reaction (about 7æl).

Heteroduplex reaction.

1. Dry the PCR products in the Speed Vac (all of the test DNAs and the normal DNA).
2. Dissolve each DNA pellet in 25æl sterile distilled water and then add:
3. 8æl 5 x HET buffer
4. 7æl labelled probe (adjust volume as necessary to give 3-5 x 105dpm)
5. Incubate for 5 min at 95oC then allow the reactions to cool down overnight by floating the tubes in the switched off water bath.

Hydroxylamine and OsO4 modifications.

NB. The hydroxylamine and OsO4 reactions should be performed in a fume cupboard.

1. For the hydroxylamine reaction proceed as follows:
   (a) Mix 1.39g Hydroxylamine with 1.6ml sterile distilled water. Adjust to pH 6.0 with diethylamine (about 1ml).
   (b) Into a sterile 1.5ml screw-capped tube add:
2. 7æl heteroduplex
3. 20æl hydroxylamine solution Incubate for 2 h at 37oC.
4. For the OsO4 modification proceed as follows: (a) Prepare a fresh OsO4 solution in a 1.5ml screw capped tube by adding:
   
5. 6.9æl 1M Tris HCl pH 8.0
6. 1.4æl 500mM EDTA
7. 41.6æl pyridine
8. 86.8æl 4% OsO4
9. 863æl Sterile distilled water Keep on ice.
   (b) Vortex to mix. The solution should be lemon yellow in colour and turbid.
   (c) Into a clean 1.5ml screw capped tube add:
10. 7æl heteroduplex
11. 18æl OsO4 solution Mix and incubate on ice for 2 h.
12. Precipitate the products of the hydroxylamine and OsO4 modifications as follows:
    (a) Add to each reaction in order:
    
13. 1æl tRNA
14. 200æl precipitation solution
15. 750æl 100% ethanol
    (b) Mix and leave in a methanol/dry ice bath for 15 min. Then centrifuge for 15 min at 14,000rpm.
    (c) Wash the pellet in 1ml 70% ethanol, centrifuge for 15 min at 14,000rpm, and Speed Vac dry.
    

Piperidine cleavage.

1. Prepare a fresh 1M piperidine solution in sterile distilled water.
2. Add 50æl of 1M piperidine to each sample, vortex and briefly spin in a microcentrifuge.
3. Incubate at 90oC for 25 min. Dry in the Speed Vac.
4. Add 200æl of sterile distilled water, mix, and dry in the Speed Vac. Repeat the wash.
5. Store the pellets at -20oC overnight.

Labelling of the 1kb ladder or HaeIII digest of í X 174.

1. Into a 1.5ml sterile screw-capped tube add:
2. 5æl 1kb ladder or í X 174 digest
3. 2.5æl Kinase buffer
4. 2æl T4 polynucleotide kinase
5. 5æl 32P -ATP (equivalent to 50æCi)
6. 25.5æl Sterile distilled water Mix and incubate at 37oC for 35 min.
7. Pass the reaction through a Nick column.
8. Dry the labelled product in the Speed Vac and redissolve in 25æl sterile distilled water. The labelled size markers (1kb ladder or í X 174 digest) are usable for several weeks. Electrophoresis of the reactions.
9. Add 20æl of stop solution to each reaction.
10. Vortex and then denature the samples at 95oC for 5 min and immediately snap cool on ice.
11. Load 2-10æl onto a 6% urea/acrylamide gel depending on the radioactivity of the sample.
12. Also load 1æl of labelled 1kb ladder or í X 174 fragments as size markers.
13. Run the gel at 2000V, 60mA, 60W, constant power, until the bromophenol blue reaches the bottom.
14. Transfer the gel to 3MM Whatman paper, cover with cling film and dry under vacuum at 80oC for 60 min.
15. Expose to Kodak XAR film at -20oC with an intensifying screen overnight.
16. Develop the autoradiograph.

6. Denaturing gradient gel electrophoresis (DGGE)

The DGGE method detects mutations (small deletions and insertions, point mutations) by separating PCR amplified DNA fragments, which differ from wild-type DNA in their melting behaviour, on a denaturing gradient gel.
The amplification products are composed of two different melting regions: a very high melting domain which is arbitrarily synthesized on the 5' end of one primer (40 bases of GC's called the GC-clamp) and a lower melting domain which could vary in length between 25 bases and about 350 bases (24). The lower melting domain is the domain of interest which is to be screened for mutations. When a fragment migrating within the gel reaches a discrete denaturing concentration, the double-stranded DNA of the lower melting domain is melted into single-strands with the effect that the amplification product will cease migrating at this position in the gel. Both strands are still connected by the higher melting domain. The position where the melting takes place within the gel depends on the base pair sequence and the base pair composition of the lower melting domain. Even when two equal double-stranded DNA fragments differ by only one base pair, they melt at different denaturing positions. A schematic diagram of this method is given in Figure 7. The separated amplification products can be made visible on an UV-transilluminator after incubating the gel with an ethidium bromide solution.
Although DGGE is a powerful method for screening DNA regions for mutations or sequence polymorphisms, substantial theoretical and practical work is required to optimize it for each DNA segment under analysis.

6.1 Preliminary theoretical work

To identify all mutations within a DNA fragment by means of the DGGE method, it is first necessary to find a unit melting domain of the region. The computer programs MELT87- and the SQHTX-program of Lerman (25,26) have been designed for this purpose. MELT87 is very helpful for finding the ideal unit melting domain of a DNA fragment of interest (Figure 8). Defining such a melting domain is also influenced by the position of the artificial GC-clamp, which could be attached to either end of the amplification product. SQHTX calculates the running distance on a gradient gel between a wild-type and a mutated DNA-fragment. The displacement is given for a helix defect for each pair in the fragment as a function of gel running time (Figure 8). Further explanation of the operation and interpretation of the two programs is supplied with the software.

6.1.1 MELT87

When the unit melting domain is determined with the computer program MELT87, the positions of the amplification primers are automatically assigned. However, in most cases an optimal melting domain is not optimal for amplification primers. Therefore, since the composition of the amplification primers is very important for good PCR results, it is often necessary to find a compromise between a useful amplification primer and a unit melting domain of an amplification product. The melting temperature (Tm) of this unit melting domain should not deviate more than 0.5oC up or down from the median temperature of its domain.
In our experience the following rules are helpful guidlines:

an effective primer is 21-26bp long (without the GC-clamp), having a well-balanced composition of bases.

Parts of one primer and parts of both primers, respectively, should not be able to hybridize against one another more than four base pairs in series.

The two external bases of one primer's end should have no more than one base complementary to the bases at the end of the other primer.

The Tm of one primer should lie between 56oC and 60oC.

Programs are available to determine the Tm of the amplification primers. Besides its effect in helping to create a unit melting domain of a DNA fragment, the GC-clamp is absolutely necessary for the gel run. When the DNA fragment has reached the point where the denaturing conditions, produced by the urea gradient, corresponds to its Tm, then the unit melting domain will melt at once into single strands. Only the GC-clamps, by keeping the single strands together as double strands in this region, fix them at this level. This short section of double-stranded DNA also allows easy visualisation of the migrated DNA fragment by ethidium bromide staining.

6.1.2 SQHTX

The values given by the program are temperature differences ( ToC) which exist between the wild-type DNA fragment and a fragment with any kind of mutation (Figure 8). The relationship between these temperature values and the denaturing concentration is 1oC = 3% denaturing concentration = 1cm distance within a 20% urea gradient gel approximately. For a given gel running time, the T (oC) temperature values for all possible mutations within the melting domain should be higher than 0.1. Otherwise it is not possible to separate the wild-type homoduplex fragment from the mutated one.

6.2 Optimising the conditions for DGGE

6.2.1 Amplification

We have found that the same basic amplification conditions can be used for each different amplification reaction. To optimize the reaction for each amplification, it is necessary only to vary the annealing temperature. This is also the only value which has to be changed when using a different thermal cycler. When using a primer with an additional sequence (GC-clamp) on its 5'- or 3'- end, better results are obtained when the time of each annealing step is longer than in an ordinary amplification reaction. In our hands 2 min annealing is sufficient.

6.2.2 DGGE

Although computer analysis is essential before one can start the practical procedure, it still takes some practical work to find the optimal running conditions. This optimisation is carried out by running several 'travel gels' which differ in the electrophoresis conditions (variation of the urea gradient and the voltage). Experience in finding the right running conditions for several different amplification products makes it easier in future work to select running conditions directly from the theoretical values since a strong correlation exists between theoretical and practical values. Use a chosen constant voltage and a given denaturing gradient (it seems reasonable to use only gradient gels, which differ by 20%) to determine the running time. If the results are not satisfactory [no distinct band(s), or retardation] choose another voltage and/or denaturing gradient. However, to find the right running conditions, change only one variable at a time. In our experience, more distinct bands are obtained when the running time is longer, rather than the voltage higher. In our laboratory best results could be obtained by using a voltage range between 25 to 60V. All three variables (voltage, running time and gradient) are dependent on the size and base content of the amplification products.
At the optimum running time the bands are well separated. In contrast to the slower migrating material a distinct pattern develops due to focusing in the gradient. In the example shown in Figure 9, the optimum running time is 20-22h (see the bands in lanes 5/6, 9/10 and 15/16.
Having established the running conditions for a DNA fragment spanning a specific gene region, these conditions can now be used to run analytical gels for mutation screening.

6.3 Practical Procedures

Full descriptions of the amplification and DGGE procedures are given in Protocol 9

The assembled equipment for DGGE is shown in Figure 10.

Protocol 9. DGGE

Equipment and Reagents

10xTAE buffer pH 7.4 (0.4M Tris-HCl, 0.2M sodium acetate, 10mM EDTA)

40% formamide deionised with a mixed bed resin and filtered through a solution filter (Schleicher & Schull)

1 x loading buffer (20% Ficoll 400, 10mM Tris-HCl pH 7.8, 1mM EDTA, pH 8.0, 0.1% orange G)

40% polyacrylamide stock solution (acrylamide:bisacrylamide = 37.5:1)

100% denaturing solution (7.0% polyacrylamide, 7.0M urea, 40% formamide, in 1 x TAE buffer pH 7.4)

0% denaturing solution (7.0% polyacryalmide in 1 x TAE buffer pH 7.4)

10% ammonium persulfate stock solution

TEMED

Ethidium bromide stock solution (10mg/ml)

electrophoresis unit (Hoefer SE 600); (glass plates 180mmx160mm; spacer thickness 0.75mm)

power supply

heating water bath with circulating pump to hold constant gel temperature (62oC) during gel run

gradient mixer in combination with a magnetic stirrer and a butterfly intravenous infusion set (a needle of outside diameter 1.9mm, connected to a tube) for pouring a denaturing gradient gel

microsyringe for loading the samples

peristaltic pump between buffer tanks to prevent significant increase in pH during electrophoresis

an UV-transilluminator for visualising gels

a documentation system (e.g. polaroid camera)

a personal computer with Lerman's software SQHTX and MELT87

Amplification

1.

Mix the following compounds for the amplification reaction (total volume of 50æl):

5æl 10 x PCR buffer

8æl dNTP-mix

500ng DNA

300ng oligonucleotide primer

900ng oligonucleotide primer with GC-clamp

2.5U Taq DNA polymerase

make up to 50æl H20

2.

Amplify according to the following conditions: Final extension 72oC 10 mins Soak 10oC

Step	Temp	Time
Denature	94oC	420 sec
Denature	94oC	35 secs
Anneal	50-57oC	120 sec 40 cycles
Extend	772oC for	60 sec + 3 secs/cycle
Extend	72oC for	600 sec

3.

Check the amplification by running 1/10th of the PCR product on a 1-2% agarose gel.

Travel schedule gels

Denaturing gradient gel electrophoresis can be carried out very successfully in a Hoefer SE 600 electrophoresis unit, attached to a circulating water bath set to a constant temperature of 62oC. All electrophoresis is performed at this temperature.

1. Preparing the appropriate solutions as follows:
2. Prepare a 40% acrylamide stock solution consisting of an acrylamide:bisacrylamide ratio of 37.5:1
3. Using this acrylamide stock solution, prepare two additional solutions:
4. a 0% denaturing solution [7.0% polyacrylamide in 1 x TAE- buffer, pH 7.4]
5. a 100% denaturing solution [7.0% polyacrylamide, 7.0 M urea, 40% formamide, in 1 x TAE buffer pH 7.4]
   Both solutions (the 0% denaturing and the 100% denaturing solution) should be prepared in large amounts, because every new preparation has a slightly different urea concentration, which changes the practical conditions of the gel run. It is necessary to reoptimise running parameters when new solutions are prepared.
6. Using these two stock solutions, prepare the two denaturing solutions (10ml each) which are needed for the gradient gel (For example, for a denaturing gradient ranging between 30% and 50% prepare a 30% and a 50% denaturing solution).
7. Prepare the travel schedule gel as follows:
8. Clean the glass plates scrupulously first with warm water, then with iso-propanol and finally with distilled water
9. Fix the glass plates within the equipment by slightly greasing the bottom of the glass plates
10. Add 5.0æl TEMED and 100æl of 10% ammonium persulphate solution to the prepared solutions for the gradient gel and pour the solutions into the appropriate chambers of a gradient mixer. Make sure that the higher concentration solution is put in the anterior chamber, while the lower concentrated solution is put in the posterior chamber. Stand the gradient mixer on a magnetic stirrer with a stirring bar inside the anterior chamber. Stir the anterior chamber fast but without producing any air bubbles. To avoid the use of a peristaltic pump, it is very important that the gradient mixer is positioned on a higher level than the glass plates within the fixation equipment. The connection between gradient mixer and glass plates is made with a "butterfly" intravenous infusion set (a needle of outside diameter 1.9mm, connected to a tube). The free end of the tube is connected to the output channel of the gradient mixer and the butterfly needle is put between the glass plates.
11. Pour the gel by opening the channel between both mixer chambers first and by opening the output channel second. Pour the gel slowly (about 5 min), as this helps to exclude air bubbles from the gel and to achieve a gradient without waves. To start the solution running within the tube, it is sometimes necessary to tap the tube.
12. Put in the comb and allow the gel to polymerise. Normally this takes about 2 h.
13. In the meantime, mix the amplification products with the loading buffer.
14. Gel electrophoresis:
15. Load a wild-type DNA sample onto the gel using 1xTAE (pH 7.4) as running buffer.
16. Begin the electrophoresis (for example 25V for a 25-45% urea/formamide gradient gel).
17. Load additional DNA samples consecutively five times with a 2h gap between each loading.
18. Stop the gel run after 24h running time.
19. Stain the gel with ethidium bromide solution and evaluate the results on a UV-transilluminator.
20. Select the optimum running time for the analytical gel.

Analytical Gel

1. Select the same gel running conditions established by the travel schedule gel (denaturing gradient, voltage and running time).
2. Pour the gel in the same manner as explained for the travel schedule gel above.
3. Form heteroduplexesa before loading the amplification products as follows: (a) Mix 10æl (80ng) of a wild-type PCR-amplified DNA with 10æl (80ng) of a patient DNA or preferably mix 10æl of two different patient DNAs (c) Heat the mixture for 5 min at 96oC in a closed water bath, switch the power off and let it cool down to room temperature overnight (b) Add sample buffer equal to one third of this volume
4. Load the DNA samples onto the analytical gel and start the gel run
5. After the gel run is completed, stain the gel with ethidium bromide for 1 h and evaluate the results on a UV-transilluminator aHeteroduplexes can also be formed using a thermal cycler as follows: denature at 96oC for 10 min, then leave at 55oC for 10 mins.

Comments on the DGGE Protocol

Heteroduplex formation improves the detection of mutations for two reasons:

it allows the simultaneous analysis of two amplification products of two different patients

it produces two additional bands and therefore allows the detection of mutations that do not change melting behaviour of the homoduplex Sometimes a point mutation results in the same base pairing, except that it is reversed (e.g. A/T T/A). In such cases, the running behaviour of the mutated and the normal DNA fragment cannot be distinguished. This problem is solved by mixing a normal and a mutated DNA sample. The formation of two additional heteroduplexes, which present now a T/T and a A/A mismatch base pair, differ much more in their melting behaviour than the original complete double-stranded DNA, thus resulting in a different running distance.
An example of the different melting behaviour of amplification products, which represents the same DNA region, but differ only in one base pair (point mutation), can be seen in Figure 11. If the base change of one DNA sample results in a completely different base pairing, we are able to identify 4 different bands: the original homoduplexes of the wild-type DNA and the patient DNA and two different heteroduplexes.
Examples of a three band pattern, where the wild-type and the mutated homoduplexes cannot be distinguished, can be seen in lanes 5 and 9 of Figure 11. If two different patient samples are used for the heteroduplex formation and a mutation pattern is found, it is not possible to decide, which patient shows the mutation. In this case, the choice is to sequence both amplification products directly or to identify the mutated DNA by mixing them each with a definitive wild-type DNA before loading onto a new analytical gel.

7. Discussion and conclusions

The three powerful screening techniques described in this chapter are able to detect all or virtually all mutations. The choice between these methods depends on a number of factors.
DGGE is useful for screening a large number of individual cases for diverse mutations in a large gene. Once the conditions have been optimised it can be rapidly used to work through very large numbers of DNA segments as was successfully demonstrated for haemophilia A (8). However, the DGGE method is not the method of choice in every case of mutation or sequence polymorphism screening since:

it take substantial effort and time to find the optimal melting domain in connection with the primer determination and to transpose the theory into practice.

DNA fragments which are about 350bp in size give good results. However, mutation screening within fragments which are larger than 400bp seems to be less sensitive and fails to identify every mutation, depending on the different migration behaviour of the mutant fragments within the gradient gel. As a consequence of this, exons (or amplification products), which are more than 400bp in size should be divided into two separate smaller overlapping amplifications.

Some genes are resistant to the DGGE method due to high G+C content, and for these an alternative screening procedure must be selected.
SSCP has the advantage of simplicity in operation although conditions need to be carefully defined to obtain optimal results. This may explain why some workers have reported rather lower sensitivity than we have found in careful comparative studies (>95%). The size of fragment screened should not exceed 350bp preferably 300bp. By multiplexing it is possible to increase throughput to approach that achievable by DGGE.
Chemical cleavage remains the standard by which other screening methods should be assessed since sensitivity approaches 100% and large fragments can be screened. However, the method is very laborious and technically demanding with requirements for toxic chemicals and a high energy emitting radiolabel. The combination of chemical cleavage with reverse transcribed transcript analysis allows large complex genes such as factor VIII or dystrophin to be screened.
However well these screening methods are performed, there remains a number of cases where no mutation is detected. Possible reasons for failure include the following:-

The wrong gene is being screened due to mis-diagnosis at the phenotype level.

The gene has been rearranged in a subtle way not detectable by exon screening. For example, about half of all cases of severe haemophilia A are due to an inversion event that leaves two halves of the gene intact but separated by several megabases (27).

The mutation causing the disease lies in an unscreened region of the gene.
Despite these difficulties and the technical demands of screening, the evident advantages of mutation specific diagnosis will probably lead to this becoming the preferred approach to genetic diagnosis in future. Large databases of mutations are accumulating for several disorders based on the results of gene screening. These have already contributed greatly to our understanding of the mechanisms of mutagenesis and the correlation between genotype and phenotype, as well as contributing to the analysis of structure and function of individual gene products. Another eventual benefit that may arise from precise identification of disease causing mutations would be the ability to repair genes through homologous recombination, as the ideal means of gene therapy. Figure 1. PCR-SSCP of exon 12 of the factor VIII gene. Clear shifts are seen in the main single-stranded doublet of lane 1. Fainter bands above the main doublet of single- stranded DNA are presumed to represent minor alternative conformations. Each of these is shifted in lane 1, confirming this interpretation. The line diagram at the base of the figure represents the PCR fragment analysed consisting of the whole of exon 12 (151bp) and 87bp and 82bp of flanking intron sequence. Lanes 1-10, patient samples; lane 11, normal DNA control; lane 12, double-stranded non-denatured DNA control. Figure 2. PCR-SSCP of a 1182 bp fragment of the 5' untranslated region of factor VIII digested with AvaII/EcoRI. Four fragments are generated, three are shown here. There is a clear shift in a single-stranded fragment of lane 10 near the 464bp double-stranded DNA. A shift in the 271bp double-stranded DNA fragment is also seen. Consecutive SSCP subsequently confirmed this shift to be from the 271bp single strands in this patient. Lanes 1-12, patient samples; lane 13, normal DNA control; ds, double-stranded non-denatured DNA control. The line diagram at the base of the figure illustrates the DNA fragments generated by restriction of the 1182bp fragment with the enzymes AvaII and EcoRI. The small hatched area represents the first 15bp of exon 1. Figure 3. Schematic representation of consecutive digestion of biotinylated PCR products in preparation for consecutive SSCP. R.E. A refers to restriction enzyme A; R.E. B refers to restriction enzyme B. Figure 4. Multiplexed SSCP analysis of the factor VIII gene. Analysis of individual normal factor VIII exons as single stranded DNA are shown in lanes 1 and 21 = exon 20, lanes 3 and 22 = exon 3, lanes 5 and 23 = exon 25 and lanes 7 and 24 = exon 16. The same normal exons 20, 3, 25 and 16 are shown as double stranded DNA in lanes 2, 4, 6 and 8, respectively. Genomic DNA samples from 12 haemophilia A patients after group 1 multiplexed analysis (see Table 1), are shown in lanes 9 to 20. Despite the complexity of the multiplexed pattern, the bands are well defined, migrate consistently and can be individually assigned to exons of origin, enabling rapid localisation of mutations. Figure 5. Schematic representation of the preparation of single-stranded DNA for direct sequencing using biotinylated oligonucleotide primers and streptavidin-coated magnetic beads. Figure 6. Chemical cleavage of exon 11 of the Factor VIII gene showing cleaved fragments in lanes 1, 2 and 3. The cleaved products in lanes 1 and 2 are identical, consistent with the same single base substitution in these patients. The cleaved product in lane 3 was due to a different missense mutation. All three mutations were detected by both hydroxylamine and OsO4. No cleavage products were detected in the samples run in lanes 4-6. Figure 7. Diagram representing a denaturing gradient gel showing the separation of two mixed wild-type DNAs (homoduplexes) and a wild-type DNA mixed with a mutant DNA (heteroduplexes). While the wild-type homoduplexes melt at one position, the heteroduplexes show 4 bands (two different heteroduplexes and the original homoduplexes) which differ in their melting behaviour. The position of the two heteroduplexes always lies above the melting point of the homoduplexes. The mutated original homoduplex may be positioned above or below the wild-type homoduplexes. At their melting point the single strands are still held together by the GC-clamp, shown as the thick line. Figure 8. Graphic demonstration of the melt map (MELT87) and the displacement map (SQHTX-program) of the factor VIII gene exon 16. The thin solid line represents the melting map [Tm(oC)] of the amplification product (exon 16 and flanking sequences) without the GC-clamp. While the x-axis indicates the base pairs of the exon 16 PCR- product, the y-axis on the left side additionally gives the melting temperature of each base pair within this amplification product. The thin line shows that the melting behaviour of the exon 16 product without the GC-clamp is very heterogenous. The thicker solid line represents the effect of an attached GC-clamp on the exon 16 PCR-product. Starting from a melting temperature of about 95oC, the line drops after 40 base pairs to a unit melting temperature of about 69oC and keeps constant over the whole amplification product. The higher melting domain in the first 40 bases represents the GC-clamp, the lower melting domain represents exon 16 and flanking sequences which can now be screened for mutations because of its unit melting behaviour. The dashed line demonstrates the displacement temperature map of factor VIII exon 16 complete with GC-clamp. The y-axis on the right side gives the different temperature values ( ToC) of each base pair within the PCR-product. These values arise, if the SQHTX-program compares the temperature differences between a possible mutation at each base pair position within a mutated fragment to the same position in a wild-type DNA duplex. The figure demonstrates, that an average temperature difference of about 0.35 and 0.40 exists over the whole of exon 16. Since we know that under practical conditions the lower limit is T 0.1oC for detecting mutations ( 0.1oC = 0.3% denaturing concentration = 0.1cm running distance in the gel), we are able to calculate that each possible mutation is detectable within exon 16. The arrows show the position of some mutations which were detected in an analytical gel. Figure 9. Travel schedule gel for the factor VIII gene exon 16. Lanes 2-7 represent a normal wild-type PCR-product which is normally used to find the right running time for the analytical gel by a chosen voltage and denaturing gradient. Samples of the same amplification product have been loaded successively from the right to the left every 2 h during the first 10 h running time (total running time = 24 h). The optimal running time for the exon 16 PCR-product is 20-22 h represented by lane 5 and 6. In these lanes retardation has already taken place (bands keep sticking on the same position) and bands are well focused compared to those in lanes 2 and 3. To demonstrate the right running conditions for an analytical gel, we have additionally loaded a mutant exon 16 amplification product (lanes 14-19) successively at the same time as the normal PCR- product from the left to the right. The different retardation compared to the wild-type fragment can be observed best after 20-22 h (lanes 5, 6 and 15, 16). In addition lanes 8-13 represent the advantage of the heteroduplex formation between a wild-type and a mutant DNA-fragment. The amplification products were also loaded from the left to the right. Besides the two different homoduplexes which are the two bands below, two additional heteroduplex bands appear, thus allowing a better mutation identification in critical cases. For further explanation see pages 38 and 39. Figure 10. Assembled equipment for DGGE. Top left, pump circulating buffer from lower to upper reservoirs of the gel electrophoresis apparatus, top right, power supply. Centre right, gel electrophoresis apparatus. Bottom right, magnetic stirrer. Bottom left, water bath set at 62oC. Centre left, pump circulating water from bath through electrophoresis tank. Figure 11. Analytical gel of the factor VIII gene exon 16 after heteroduplex formation of patient DNA-samples with a normal DNA. Mutations can be seen by identification of additional bands above the normal band (see lanes 3-9). Controls consisting of heteroduplexes formed between two normal DNAs, are in lane 2 and 10. Lane 1 and 11 contain a HindIII digest of í x 174 DNA as size markers.