Molecular and Cytogenetic Analysis

Reagents

Method

Optimal conditions for the reaction have to be derived empirically, with the magnesium concentration and annealing temperature being the most important parameters. The choice of buffer depends on the enzyme being used, and the company will usually supply the most appropriate one. For genes with a high GC content, buffer II in combination with 10% DMSO may give better amplification. In most cases, a 25-μl reaction volume suffices. A no template control (NTC) should always be included (i.e. a reaction without any DNA or complementary DNA [cDNA]: the template) to control for contamination. If the NTC yields a product, the analysis is invalid. A DNA sample that is known to amplify can also be included and this sample may then be used as a positive control.

The risk of contamination cannot be over emphasised. This can be minimised by using plugged tips and having dedicated micropipettes and areas for each step of the analysis. The optimum cycling conditions need to be determined for each thermocycler. Specificity is often improved by ‘hot start’ PCR. This is achieved by setting up all the PCR tests on wet ice and transferring the tubes to the thermocycler once it reaches 95 °C or by using an enzyme that only becomes activated when heated at 95 °C for several minutes. In preparing a group of reactions, a premix solution is prepared that can be dispensed into microcentrifuge tubes, tube strips or PCR reaction 96-well plates to which the template DNA is added. When a particular PCR is to be performed repetitively over a period of time, it is helpful to prepare a large volume (e.g. 10 ml) of the reaction mixture (without DNA or Taq polymerase), aliquot it in amounts sufficient for 20 reactions and store it at − 20 °C.

• 1.

  Prepare a PCR mixture for 20 reactions (with a final volume of 25 μl for each DNA sample) as follows:

  Stock Solution	Vol (μl)	Final Concentration
  × 10 PCR buffer III	50	× 1
  25 mmol/l MgCl 2	40	2.0 mmol/l
  10 mmol/l dNTP	10	0.02 mmol/l
  10 μmol/l Primer (1)	20	0.04 μmol/l
  10 μmol/l Primer (2)	20	0.04 μmol/l
  5 u/μl Taq polymerase	2	0.02 μ/ml
  Water	358
  Final volume	500

  Add the Taq polymerase last, mix well and pulse-spin in a microcentrifuge to bring down the contents of the tube.

• 2.

  Aliquot 24 μl of the PCR reaction mix into each tube. Add 1 μl of template DNA at approximately 0.05 mg/ml into all bar one (the NTC) of the reaction tubes.

• 3.

  Place the tubes in a PCR machine, using a heated lid, programmed for the following conditions: an initial step of 5 min at 95 °C and then 30 cycles of 95 °C for 45 sec, 58 °C for 45 sec, and 72 °C for 1 min in sequence followed by a final extension step at 72 °C for 10 min. These conditions are suitable for many primer pairs, although some will require different annealing temperatures or longer extension times. These two parameters need to be optimised in advance.

• 4.

  While the PCR program is running, a 1.5% agarose minigel is prepared: add 0.75 g of agarose to 50 ml of × 0.5 TBE buffer and heat until completely dissolved. Add 2 μl of Sybr Green, allow the agarose to cool slightly and pour with the appropriate comb in position.

• 5.

  To check if the amplification has been successful, add 1 μl of tracking dye to a 10-μl aliquot of the PCR reaction mixture, being careful not to pipette the mineral oil overlaying the PCR reaction.

• 6.

  Load the gel and run at a constant voltage of 100 V for 1 hour in × 0.5 TBE buffer. A molecular size marker should be included to establish the size of the amplified fragment; these are commercially available.

• 7.

  Visualise the DNA on an ultraviolet (UV) transilluminator and take a photograph.

Presence or absence of a PCR product and sizing PCR products

Initially, PCR products were commonly visualised by agarose gel electrophoresis or using a fast automated system such as Qiaxcel equipment (Qiagen). However, it has also become commonplace to visualise PCR products directly on DNA analysers – in particular the Applied Biosystems 3130xl, 3500 or similar models ( www3.appliedbiosystems.com ). These read fluorescently labelled DNA fragments as they exit from the gel and enable single-base resolution from around 50–800 bp. If appropriate primers and controls are included in an experiment, the actual presence of a product can be highly informative.

Deletions and insertions can be easily identified when the size of the PCR product significantly differs from the size in the normal control. An example of this analysis is given for α globin gene Gap analysis on page 136.

Higher resolution of fragment sizes is obtained by capillary electrophoresis, which is particularly appropriate in the analysis of short tandem repeat (STR) sequences, which can be highly variable in length and therefore useful as genetic markers of different individuals. High resolution sizing of DNA fragments is now often performed on DNA analysers as mentioned above. Several examples of these applications are given in this chapter, in particular in the section describing chimaerism analysis on page 159.

Principle

Point mutations and small insertions or deletions can be identified directly by the presence or absence of a PCR product using allele-specific primers. ^, Two different oligos are used that differ only at the site of the mutation (the amplification refractory mutation system, or ARMS, primers) with the mismatch distinguishing the normal and mutant base located at the 3′ end of the oligo. An oligo with a mismatch at its 3′ end will fail to prime the extension step of the reaction. Each test sample is amplified in two separate reactions containing either a mutant ARMS primer or a normal ARMS primer. The mutant primer will prime amplification in combination with one common primer from DNA with this mutation but not from a normal DNA. A normal primer will do the opposite. To increase the instability of the 3′ end mismatch, and so prevent the failure of the amplification, it is sometimes necessary to introduce a second nucleotide mismatch three or four bases from the 3′ end of both oligos. A second pair of primers located at a distance from the ARMS primers is also included in the reactions as an internal control for the efficient amplification of DNA. This is essential because a failure of the ARMS primer to amplify is interpreted as a significant result and must not be the result of suboptimal amplicon reaction.

Interpretation

The fragment produced by amplification with the internal control primers must be seen in all the samples, except for the NTC. Then the presence or absence of a mutation is simply determined by the presence or absence of the amplification with the mutant ARMS primer. Similarly, the presence or absence of the normal allele is determined in the same way with the normal ARMS primer. In this way, heterozygous, homozygous normal, and homozygous mutant genes can be distinguished. An example of the application of this technique will be presented with the diagnosis of β thalassaemia mutations on page 135.

Reagents

A number of companies now supply a comprehensive range of restriction enzymes (REs), but they may vary greatly in their cost. Those that are in regular use are generally quite inexpensive compared with the more specialised enzymes that are used only occasionally and that may be 10–100 times more expensive. RE buffers are now almost always supplied with each RE. Buffer compositions are always given and will vary from enzyme to enzyme. Many commonly used REs cut perfectly well in a single ‘universal’ buffer. This is prepared using the following stock solutions:

• 1.

  Tris-acetate, 2 mol/l, pH 7.5. Dissolve 24.2 g of Trizma base in 60 ml of water, adjust the pH to 7.5 with glacial acetic acid and make up to 100 ml.

• 2.

  Potassium acetate, 2 mol/l. Weigh out 19.62 g, make up to 100 ml with water and dissolve.

• 3.

  Magnesium acetate, 2 mol/l. Weigh out 42.89 g, make up to 100 ml with water and dissolve.

• 4.

  BSA fraction V (molecular biology grade), 20 mg/ml .

• 5.

  Dithiothreitol (DTT), 0.5 mol/l. Weigh out 0.771 g, make up to 10 ml with water, dissolve and store at − 20 °C.

• 6.

  Spermidine (N-(3-aminopropyl)-1,4-butane-diamine), 1 mol/l. Weigh out 1.273 g, make up to 10 ml with water, dissolve and store at − 20 °C.

• 7.

  × 10 RE buffer. For a × 10 concentrated buffer, prepare a solution that is 300 mmol/l Tris-acetate, pH 7.5, 660 mmol/l potassium acetate, 100 mmol/l magnesium acetate, 1 mg/ml BSA, 10 mmol/l DTT, and 30 mmol/l spermidine; aliquot into microcentrifuge tubes and store at − 20 °C.

Method

• 1.

  To a 20-μl aliquot of a PCR product add 2.5 μl of × 10 restriction enzyme buffer, 2 μl of double-distilled water, and 2–5 units of the appropriate RE (usually 0.5 μl), giving a final volume of 25 μl. In preparing more than one digestion with the same restriction enzyme, sufficient buffer, enzyme, and water can be premixed and dispensed into the PCR products.

• 2.

  Incubate at 37 °C (or other temperature as specified by the manufacturer) for a minimum of 4 h.

• 3.

  Pour a 2.5% agarose minigel in a taped casting tray with the appropriate comb. The gel is made up of 1:1 mixture of type II medium electroendosmosis agarose and NuSieve agarose ( www.thermofisher.com ) (i.e. 0.675 g of agarose and 0.675 g of NuSieve agarose in 50 ml of half-strength [× 0.5] TBE buffer).

• 4.

  After the incubation period, add 2 μl of tracking dye to the digests and load the samples on to the gel. The electrophoresis is continued until a clear separation of all the expected fragments is achieved, which may be checked at intervals by placing the gel on a UV transilluminator.

Principle

The Sanger chain termination method for direct DNA sequencing has become a standard diagnostic tool. In many laboratories this procedure has superseded targeted mutation detection as it provides a robust and relatively rapid method to identify all sequence changes that may be present in a particular DNA fragment. This approach is particularly relevant where multiple different mutations may underlie a particular disorder. This is the case for β thalassaemia and for glucose-6-phosphate dehydrogenase (G6PD) deficiency, so it is not surprising that DNA sequencing has often become the method of choice for the molecular diagnosis of these disorders.

The method involves the de novo synthesis of DNA strands in one direction from a PCR-derived template amplicon. The chain is lengthened by a thermostable DNA polymerase using deoxynucleotide triphosphates in the normal way. However, included in the reaction mixture is a small proportion of fluorescently labelled dideoxynuclotide triphosphates (ddNTPs), which when incorporated will prevent any further extension of the chain. This process happens millions of times along a relatively short piece of DNA (typically 30–400 and no more than 800–1000 bases), which means that chain-termination will occur many times at each position along the fragment. In the ABI BigDye system (Life Technologies, www.thermofisher.com ), each of the ddNTPs is labelled with a different fluorochrome, and so the products of the sequencing reaction will consist of single-stranded DNA fragments, each differing in size by one base pair and each labelled with a different colour. These fragments can then be separated by capillary electrophoresis according to size, and the order with which the different colours exit from the capillary will correspond to the sequence of the DNA template.

Methodology

Interpretation

Reading the DNA sequence from a trace – known as an electrophoretogram – is relatively straightforward as long as the quality of the trace is satisfactory: an A is shown as a green peak, T as a red, C as a blue and G as a black. Free software packages are available for viewing these traces, and indicate the DNA sequence in the file. Simple alignment of this sequence to the GenBank reference sequence can be performed at the National Center for Bioinformatics (NCBI) ( www.ncbi.nlm.nih.gov ) using the Blast program, and will identify any sequence changes. Heterozygous point mutations will be seen as double peaks, with two colours overlaid. Small heterozygous insertions or deletions (indels) are harder to decipher, as the sequence 3′ of the mutation will be a double sequence, with the reference and indel-variant allele superimposed on one another: the extent of the indel can be defined by subtracting from the expected normal sequence.

Principle

High resolution melt (HRM) curve analysis is a post-PCR analytical methodology used for identifying genetic variants in suitable regions of interest in candidate genes. Once optimised, it is simple and fast with no post-PCR manipulation. Analysis is performed using Applied Biosystems High Resolution Melt Software ( www.appliedbiosystems.com ).

HRM analysis is performed on double-stranded (ds) DNA samples. Firstly the DNA is amplified using a real-time platform prior to the HRM melt phase. The HRM process is a slow denaturation of the dsDNA from 50–95 °C in conjunction with an intercalating fluorescent dye, SYTO9 ( www.thermofisher.com ). When the melting temperature of the ds DNA is reached, the two strands ‘melt’ apart. The midpoint of the melt curve is described as the point when 50% of the DNA is double stranded and 50% is single stranded. The shape of the curve is dependent upon the characteristics of the ds DNA, which relate to whether it is homozygous wild-type, homozygous mutant or heterozygous wild-type and mutant. When the two strands ‘melt’ apart the fluorescence level drops. As the HRM is monitored in real-time, this curve gives a real time picture of the characteristics of the DNA being tested.

The lower limit sensitivity of HRM, as determined by our laboratory using the ABI3100 instrument ( www.appliedbiosystems.com ), is approximately 10%. However, this is dependent upon the mutation being tested as sensitivity is partly determined by the base change. The greater the temperature shift, the more sensitive the test will be.

Methodology

Interpretation

Homoduplexes, whether wild-type or mutant, will display the same curve shape but will be distinguished primarily by the shift on the temperature axis ( x axis). Heteroduplexes are discriminated by a change in the shape of the curve.