There are several types of mutation that can occur in nucleic acids, either transiently or those that are stably incorporated into the genome. During evolution, mutations may be inherited in one or both copies of a chromosome, resulting in polymorphisms within the population. Mutations may potentially occur at any site within the genome; however, there are several instances whereby mutations occur in limited regions. This is particularly obvious in prokaryotes, where elements of the genome (termed hypervariable regions) undergo extensive mutations to generate large numbers of variants, by virtue of the high rate of replication of the organisms. Similar hypervariable sequences are generated in the normal antibody immune response in eukaryotes. Mutations may have several effects upon the structure and function of the genome. Some mutations may lead to undetectable effects upon normal cellular functions, termed conservative mutations. An example of these are mutations that occur in intron sequences and therefore play no part in the final structure and function of the protein or its regulation. Alternatively, mutations may result in profound effects upon normal cell function, such as altered transcription rates or on the sequence of mRNAs necessary for normal cellular processes.

Mutations occurring within exons may alter the amino acid composition of the encoded protein by causing amino acid substitution or by changing the reading frame used during translation. These point mutations were traditionally detected by Southern blotting or, if a convenient restriction site was available, by restriction fragment length polymorphism (RFLP). However, the PCR has been used to great effect in mutation detection since one can amplify the desired region of DNA with a common sequence (e.g. M13) embedded within one of the primers and perform Sanger sequencing. A more high-throughput approach is the allele-specific oligo nucleotide PCR ( ASO-PCR) where two competing primers and one general primer are used in the reaction (Figure 1). One of the primers is directly complementary to the known point mutation, whereas the other is a wild-type primer; that is, the primers are identical except for the terminal 3′ end base. Thus, if the DNA contains the point mutation, only the primer with the complementary sequence will bind and be incorporated into the amplified DNA, whereas if the DNA is normal, the wild-type primer is incorporated. The results of the PCR are analysed by agarose gel electrophoresis. A further modification of ASO-PCR has been developed where the primers are each labelled with a different fluorophore. Since the primers are labelled differently, a positive or negative result is produced directly without the need to examine the PCRs by gel electrophoresis.

Fig1. Point mutation detection using allele-specific oligonucleotide PCR (ASO–PCR).

Various modifications now allow more than one PCR to be carried out at a time ( multiplex PCR ), and hence the detection of more than one mutation is possible at the same time. Where the mutation is unknown it is also possible to use a PCR system with a gel-based detection method termed denaturing gradient gel electrophoresis (DGGE). In this technique, a sample DNA heteroduplex containing a mutation is amplified by the PCR, which is also used to attach a GC-rich sequence to one end of the heteroduplex. The mutated heteroduplex is identified by its altered melting properties through a polyacrylamide gel that contains a gradient of denaturant such as urea. At a certain point in the gradient the heteroduplex will denature relative to a perfectly matched homoduplex and thus may be identified. The GC clamp maintains the integrity of the end of the duplex on passage through the gel (Figure 2). The sensitivity of this and other mutation detection methods has been substantially increased by use of the PCR, and further mutation techniques used to detect known or unknown mutations are indicated in Table 1. An extension of this principle is used in a number of detection methods employing denaturing high-performance liquid chromatography (dHPLC). Commonly known as wave technology, the rapid detection of denatured single strands containing mismatches allows a high-throughput analysis of samples to be achieved.

Fig2. Detection of mutations using denaturing gradient gel electrophoresis (DGGE).

Table1. Main methods of detecting mutations in DNA samples

Polymorphisms are particularly interesting elements of the human genome and as such may be used as the basis for differentiating between individuals. All humans carry repeats of sequences known as minisatellite DNA , of which the number of repeats varies between unrelated individuals. Hybridisation of probes that anneal to these sequences using Southern blotting is one method to type and identify those individuals; there are also PCR methods for mini/microsatellites.

Multiplex ligation-dependent probe amplification ( MLPA) is a PCR-based multi plex assay that allows a number of target sites, such as deletions, duplications, mutations or SNPs, to be amplified with a pair of primer-containing probes ( Figure3). The process involves a denaturation and hybridisation stage, a ligation stage and a PCR amplifi cation stage, followed by analysis of the products. The technique involves designing two adjacent hybridisation probes that contain the fl uorescence-labelled forward sequence and the reverse primer sequence used in the amplification stage. The latter is designed with a stuffer sequence that can be varied to suit the target. The probes are first hybridised to the denatured sample DNA. In the next stage, the two oligonucleotide probes are joined by ligation – only ligated probes can be used in the amplification stage. Oligonucleotide probes that are not ligated will only contain one primer sequence, and as a consequence cannot be amplified and generate a signal.

Fig3. Multiplex ligation-dependent probe amplification (MLPA). In this method, two hybridisation probes are used, one containing a forward PCR primer sequence labelled with a f luorescent dye, the other with a stuffer sequence and reverse primer sequence. Denaturation and hybridisation with the target sequence takes place, after which a ligation step is employed. If ligation takes place then PCR amplification can proceed; however, if no ligation occurs PCR does not take place and thus no target DNA is present.

DNA fingerprinting is the collective term for two distinct genetic testing systems that use either ‘ multi-locus’ probes or ‘ single-locus’ probes. Initially described DNA fingerprinting probes were multi-locus probes and so termed because they detect hypervariable minisatellites throughout the genome, i.e. at multiple locations within the genome. In contrast, several single-locus probes were discovered that under specifi c conditions only detect the two alleles at a single locus and generate what have been termed DNA profiles because, unlike multi-locus probes, the two-band pattern result is in itself insufficient to uniquely identify an individual.

Techniques based on the PCR have been coupled with the detection of minisatellite loci. The inherent larger size of such DNA regions is not best suited to PCR amplification; however, new PCR developments are beginning to allow this to take place. The discovery of polymorphisms within the repeating sequences of minisatellites has led to the development of a PCR-based method that distinguishes an individual on the basis of the random distribution of repeat types along the length of a person’s two alleles for one such minisatellite. Known as minisatellite variant repeat (MVR) analysis or digital DNA typing , this technique can lead to a simple numerical coding of the repeat variation detected. Potentially, this combines the advantages of PCR (sensitive and rapid) with the discriminating power of minisatellite alleles. Thus for the future, there are a number of interesting identification systems under development and evaluation. Techniques for genetic detection of polymorphisms have been used in many cases of paternity testing and immigration control, and are becoming central factors in many criminal investigations. They are also valuable tools in plant biotechnology for cereal typing and in the fi eld of pedigree analysis and animal breeding.

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مواضيع ذات صلة

Microarrays (DNA Chips)

The Largest Subunit in RNA Polymerase II Has an Essential Carboxy-Terminal Repeat

Three Eukaryotic RNA Polymerases Catalyze Formation of Different RNAs

Regulatory Elements in Eukaryotic DNA Are Found Both Close to and Many Kilobases Away from Transcription Start Sites

Control of Gene Expression in Bacteria

Nucleic Acid Electrophoresis

Analysing Genes and Gene Expression

Screening Gene Libraries

Hybridisation and Gene Probes

Cloning Vectors: Vectors Used in Eukaryotes

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes

Centromere Sequences Vary Greatly in Length and Complexity