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MUTATION DETECTION

The field of clincal molecular diagnostics has grown rapidly over recent years as an increasing number of genes associated with specific diseases have been discovered and charecterised. Gene mutations have been defined for many inherited diseases and increasingly sophisticated tests for mutations in genomic DNA have being developed and are now used in routine diagnostic practice. Many different techniques have evolved for this purpose but they all share the goals of effectively and efficiently detecting mutations which are significant for specific diseases while remaining cost effective, simple and easy to use and allow the required level of sample throughput.

Nucleotide sequencing remains the gold standard for the detection and definition of gene mutations as both functions are unequivocally performed at the same time. However, there are a range of other methodologies which are used to either detect the presence of specific pre-defined nucleotide sequences or to quickly scan for all mutations present in large stretches of gene sequence more efficiently than sequencing. Sequencing is still widely used as a reference technique to confirm mutations highlighted by other methods.

Some of the most commonly used techniques for the detection of mutations in human genomic DNA are described below.

Mutation Scanning Methods
RFLP - Restriction Fragment Length Polymorphism

This is probably the simplest and cheapest of all mutation scanning techniques. Insertions and deletions (and occasionally single base changes) in genomic DNA can be detected by cutting the DNA with restriction enzymes and simply observing the pattern of bands generated after gel electrophoresis. Restriction enzymes are carefully selected to give a suitable spread of fragment sizes on the gel. This can be done directly on genomic DNA without further template amplification and may be combined with southern blotting to enhance sensitivity and specificity.

Fragment Length Analysis

A related fragment length analysis technique involves amplifying small (approximately 400bp) fragments of genomic DNA by PCR instead of performing restriction enzyme digests. Single base insertions and deletions can be resolved with this technique when the fragments are separated by PAGE.

Physical Methods

All these methods rely on the physical properties of nucleic acids and involve alterations in their mobility during gel electrophoresis. These assays are generically referred to as mobility shift assays.

Heteroduplex Analysis

All variations of this method rely on the effect of a conformational change on electrophoretic mobility induced by mismatched bases in a heteroduplex formed between a wild type (normal) control strand and test (either normal or mutant) strand. The mismatched bases are thought to cause bulges, bends or rotations in a DNA heteroduplex that causes it to migrate more slowly through a gel than duplexes between totally complimentary strands of the same sequence. These techniques can theoretically detect all the mutations (including point mutations) in a particular nucleotide sequence although, in practice, detection rates are usually in the range of 75 to 100%.

SSCP - Single Strand Conformation Polymorphism (also called SSCA Single Strand Conformation Analysis)

This relatively new technique (introduced in 1989) is based on the sequence specific differential mobility of single-stranded nucleic acid (DNA or RNA) molecules in a non-denaturing gel. The principle is that when a single-stranded nucleic acid is placed in a non-denaturing solution it will fold in a sequence specific manner. Introduction of a mutation will alter the folding and this differential folding leads to differential electrophoretic mobility. Wild type (normal) control strands and test (either normal or mutant) strands are run side by side on a gel and differences in mobility interpreted as the presence of sequence differences (mutations). The fact that there are two complimentary strands gives each mutation two chances of being detected and 50 to 100% detection rates have been quoted. Detection of 100% of mutations may require running several gels with differing non-denaturing conditions.

DGGE - Denaturing Gradient Gel Electrophoresis

These methods utilise the sequence specific differential melting characteristics of duplex DNA in a temperature or chemical gradient. When a DNA duplex is treated to an increasing denaturation gradient the two strands begin to melt. The strands do not separate all at once. The AT rich regions melt first, as they are held together less strongly than the GC rich regions. Duplexes of different sequences that are run side by side on a gel, across an increasing temperature or chemical gradient, will migrate at different speeds depending on the relative degree of strand separation.

Chemical and Enzyme Cleavage of Mismatch (CCM and ECM)

These techniques are based on the ability of enzyme or chemical treatments to selectively cleave heteroduplexes between mutant (test) and wild type (control probe) DNA strands at the site of every mismatch. The enzyme based methods utilise various enzymes which recognise and cleave the duplex at each mismatched base and the chemical methods are based on piperidine-mediated cleavage of chemically modified T and C mismatched bases. The presence of mismatches in the heteroduplexes and therefore mutations in the test DNA can be inferred from the pattern of bands detected on the gel.

The Protein Truncation Test (PTT) is different from most other mutation detection methods in that it detects mutations at the protein level rather than the DNA level. It detects only those mutations which lead to truncation of the normal protein product and which almost certainly degrade the normal function of that protein. An RNA polymerase promoter region and translational start codon are introduced by PCR amplification into nucleic acid sequences extracted from a clinical sample. The protein encoded by the PCR product is then expressed during an in vitro transcription and translation reaction and the resulting products are separated by PAGE. Mutations leading to the formation of an internal stop codon give rise to a shorter than expected protein product. Only translation termination mutations are detected.

Microsatellite Instability Assays or Short Tandem Repeat (STR)

This potential diagnostic technology relies on detecting instability in regions of repeated genomic DNA (microsatellites or tandem repeats). This kind of instability is thought to be indicative of problems in the cells mismatch repair mechanisms. Mismatch repair errors have been linked to colon cancers and carcinoma of the head and neck. Only between 12 to 60% of patients with replication error associated tumours actually exhibit microsatellite instability. Unstable trinucleotide repeats have also been implicated in genetic diseases such as Fragile X Syndrome, Myotonic Dystrophy (MD), Huntington's Disease (HD) and Spinal Bulbar Atrophy (SBA). Detection of the number of repeats (variation of which indicates instability) at a specific disease loci is estimated by the relative mobilty of PCR products amplified from that loci compared to allelic ladders or other molecular weight markers.

Also referred to as the Sequence Specific Oligonucleotide (SSO) method it is based on the principle that conditions can be found that allow binding of a perfectly complementary labelled oligonucleotide to a specific pre-defined template sequence but not to a template that differs by only one nucleotide. Large quantities of template DNA are amplified by PCR from genomic DNA, fixed to a membrane and probed with numerous wild type and mutant probe pairs. Probes are often labelled with biotin and binding events detected with the use of streptavidin conjugated enzyme systems (Horse Radish Peroxidase or Alkaline Phosphatase).

OLA - Oligonucleotide Ligation Assay

This technique is based on the principle that a ligase enzyme can join together two oligonucleotides that are bound to adjacent regions on a complementary stretch of template DNA. Conditions can be found that prevent this ligation if the abutting base at the 3’ end of one of the oligonucleotides is mismatched and therefore not bound to the template strand. In other words, ligation between the oligonucleotides only takes place if they are both complementary along their entire length to the template DNA. Detection methods are employed to differentially detect the ligation products separated by gel electrophoresis and numerous pairs of oligonucleotides with suitably varying electrophoretic mobility can be used to detect multiple defined mutations at the same time.

In one variation of this technique, differentially labelled oligonucleotides are included which are complementary to both the wild type and mutant sequences. A ligation product is therefore always produced and the molecular weights of the wild type or mutant oligonucleotides are engineered such that their ligation products migrate at different rates during gel electrophoresis and are easily distinguished.

Allele-Specific PCR

This technique is conceptually very straight forward and is based on the principle that 3’ mismatches in one of a pair of PCR primers will prevent PCR amplification. A common primer is combined with a primer designed to be complementary to either the wild type or mutant sequence. The presence or absence of the mutation is determined by whether or not amplification takes place. Recent advances in detection technology have allowed the detection of PCR products to be done in real time as the amplification reaction takes place without the need to run a gel or even open the vessel in which the reaction takes place. The two commercial versions of this idea involve the use of fluorescent probes.

An increasing number of genes have been identified in which a significant proportion of the disease causing mutations associated with that gene result in premature termination of protein translation. Promega is a major supplier of the reagents used in a rapid and efficient test, designated the Protein Truncation Test (PTT), which fascilitates identification of this type of translation termination mutation. Although initially developed for use with Duchenne Muscular Dystrophy (DMD), PTT is now widely used for screening many different genes associated with various diseases.

The Protein Truncation Test (PTT) allows mutation detection at the level of protein expression rather than at the level of genomic DNA sequence. PTT detects only mutations which cause premature termination of translation and this type of mutation has been linked to many diseases, including breast, ovarian and colon cancer.

1. Nucleic acid isolation from blood or tissue.
2. Amplification of the gene segments by PCR (or, when investigating mRNA, by RT-PCR).
3. Transcription/translation of the PCR product in a coupled reaction using the TNT® T7 Quick Coupled Transcription/Translation System.
4. Separation of the proteins by SDS Polyacrylamide Gel Electrophoresis (SDS Page).

The presence of a premature termination codon is indicated by the visualisation of a protein which is smaller in molecular weight (and therefore runs faster through the gel) than the wild type protein. Detection of the translated proteins is performed by either radioactive or non-radioactive methods.

• A single PTT reaction allows the analysis of large gene fragments (2-4kb).
• PTT detects mutations of pathological interest (ie those that lead to truncated proteins). Phenotypically silent mutations are not detected.

• The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorised or unlicenced use of the PCR process. Use of this product is recommended for persons that either have a licence to perform PCR or are not required to obtain a licence.

AMPLIFICATION

Amplification of nucleic acids is an integral part of many molecular diagnostic assays and diverse variations of this technique now exist including routine PCR*, RT-PCR, high fidelity PCR and hot start PCR.

Promega is committed to your success in PCR and by using our reagents you can be sure you have the best tools to achieve optimal performance in your reactions.

Look for the Promega PCR satisfaction guarantee on all our amplification products.

RT-PCR is the combination of a reverse transcription reaction with a polymerase chain reaction (PCR) to allow amplification of a small number of RNA molecules into many copies of double-stranded DNA. This technique can be used to determine the presence or absence of a transcript, to estimate expression levels and to clone cDNA products without the necessity of constructing and screening a cDNA library.

A number of factors should be considered when selecting the optimal system for RT-PCR. Important considerations include the optimal temperature for reverse transcription, the level of sensitivity required, the downstream applications for the PCR product and ease of use. Three of the most common solutions for performing an RT-PCR reaction are outlined in the following flow diagram.

RNA PCR Kit RNA PCR Kit RT-PCR Kit

Assemble Master Mix M-MLV Tth AMV and Tfl

Synthesise First Strand 42° C, 45 minutes 60° C, 45 minutes 48° C, 45 minutes

Inactivate Reverse 99° C, 5 minutes Not applicable Not applicable

Transcriptase

Add further Reagents Taq, primer Not applicable Not applicable

and buffer

Denature Template 94° C, 2 minutes 94° C, 2 minutes 94° C, 2 minutes

Synthesise Second 40 cycles 40 cycles 40 cycles

Strand and Amplify DNA

Number of steps 6 4 4

Number of reactions 2 1 1

Sensitivity +++ + +++

Simplicity + + +++

Promega’s Access RT-PCR System is a one tube, two-enzyme system and provides the most sensitive, quick and reproducible analysis of even rare RNAs. The system uses AMV Reverse Transcriptase (AMV RT) from Avian Myeloblastosis Virus for first strand cDNA synthesis and the thermostable Tfl DNA Polymerase from Thermus flavus for second strand cDNA synthesis and DNA amplification. The Access RT-PCR System includes an optimised single-buffer system that permits extremely sensitive detection of RNA transcripts, without a requirement for buffer additions between the reverse transcription and PCR* amplification steps. This simplifies the procedure and reduces the potential for contaminating the samples. In addition, the improved performance of AMV Reverse Transcriptase at elevated temperatures (48°C) in the AMV/Tfl 5X Reaction Buffer minimises problems encountered with secondary structures in RNA.

* The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorised or unlicenced use of the PCR process. Use of this product is recommended for persons that either have a licence to perform PCR or are not required to obtain a licence.

Male Infertility and Microdeletions in the Y-Chromosome.

It has been estimated that 15% of all couples are affected by infertility and that the male is responsible for at least 40% of these cases. He may also share responsibility with his partner in an additional 20%. Two percent of men have infertility related to severe defects in sperm production and it has been suggested that up to 20% of all severe male-factor infertility cases are associated with microdeletions in the Y-chromosome.

Identifying and characterising deletions in the Y-chromosome is the focus of a number of ongoing investigations into the causes of male infertility.

Available from July 1999

Promega has developed a rapid PCR** based method for the detection of 18 unique and specific Y-linked genomic deletions. The Y-Chromosome Deletion Detection System Version 1.1* provides a standardised screening panel amplifying only informative non-polymorphic sequence tag sites (STS) on the q arm of the Y chromosome. The system amplifies key functional regions – associated with the AZoospermia Factor (AZF), including regions which flank AZFa and cover AZFb, AZFc, AZFd plus DAZ, Kal-Y, SMCY and flanking loci for key spermatogenesis related genes (namely RBM1, DFFRY, and DBY ).

This system utilises a total of 18 Y-specific primer pairs which have been combined into four multiplex master mixes to reduce the number of separate amplification reactions needed per sample. Each multiplex also contains an X-specific primer pair (SMCX) which is included as an internal amplification control. The amplification products (80-400bp) of the four multiplex PCR** reactions are readily separated by agarose gel electrophoresis and easily visualised by ethidium bromide staining. The system includes the controls needed to ensure the integrity of the amplification reactions and a molecular weight ladder to minimise deletion analysis time and the possibility of misinterpreting band molecular weights.

Failure to amplify specific regions of the Y- chromosome is indicative of Y-chromosome deletions in the test sample

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