What is SSCP?
SSCP Analysis:Single-Strand Conformation Polymorphism Analysis
SSCP is the simplest and most used method of mutation detection. PCR is used to amplify the region of interest and the resultant DNA is separated as single-stranded molecules by electrophoresis in a non-denaturing polyacrylamide gel (Orita et al, 1989). A strand of single-stranded DNA folds differently from another if it differs by a single base, and it is believed that mutation-induced changes of tertiary structure of the DNA results in different mobilities for the two strands. These mutations are detected as the appearance of new bands on autoradiograms (radioactive detection), by silver staining of bands or the use of fluorescent PCR primers which are subsequently detected by an automated DNA sequencer (non-radioactive detection).
The tertiary structure of single stranded DNA changes under different physical conditions e.g. temperature and ionic environment. Hence the sensitivity of SSCP depends on these (and many other) conditions (see below). Whilst some empirical rules have emerged for the choice of separation conditions for sequence variants in particular sequence contexts, it is not possible to predict whether a certain mutation can be detected under given conditions, especially when the mutation is in a new sequence context. Mutation detection for PCR-SSCP is generally high, >80% in a single run for fragments shorter than 300bp (Hayashi and Yandell, 1993). As sensitivity is not 100%, the absence of a new band does not prove that there is no mutation in the analysed molecule.
The sensitivity of PCR-SSCP decreases with increasing fragment length, <300bp being the optimum. For mutation detection in longer fragments (exons >300bp and whole cDNAs) overlapping short primer sets can be used, or long PCR products digested with appropriate restriction enzymes prior to SSCP (however, reamplification of individual new bands with the original primer set is now no longer possible).
SSCP screening has two primary advantages as a mutation-screening technique:
The only step necessary after PCR amplification is a heat denaturation in formamide and NaOH.
SSCP screening only tells you that a mutation exists. You must perform subsequent DNA sequencing to determine the nature of the mutation that caused an electrophoretic mobility shift in a given sample.
Moreover, not all point mutations in a given sequence will cause a detectable change in electrophoretic mobility. However, by optimizing PCR reactions and run conditions before attempting a large-scale screening you can increase the sensitivity.
Variations and modifications of SSCP:
REF-SSCP: Restriction endonuclease fingerprinting. A modification of SSCP where a 1-kb segment is digested with 5 restriction enzymes designed to produce fragments of ~150bp (Factor IX gene). After digestion the products are mixed, end-labelled with 32P, denatured and electrophoresed under non-denaturing conditions. Two 'components' are evident; the gain or loss of restriction site 'informative restriction component' or abnormal mobility of the 5 restriction fragments (10 strands) called the 'SSCP component'. Efficiency of detection: 96% detection (5.6%polyacrylamide/23oC) or 100% (7.5%GeneAmp/23oC or 8oC) of 24 test mutations (Liu and Sommer, 1995).
Introduction to Microsatellite Analysis
What is Microsatellite Analysis?
Microsatellite loci are PCR amplified and the PCR products are then analyzed by electrophoresis to separate the alleles according to size. PCR-amplified microsatellite alleles can be detected using various methods, such as fluorescent dye labeling, silver staining, or fluorescent dye staining.
The number of repeat units at a microsatellite locus may differ, so
alleles of many different lengths are possible. Microsatellite loci occur
throughout the genome of most organisms and therefore have been used as
markers to establish linkage groups in crosses and to map genetically
identified mutations to chromosomal positions.
The lengths of microsatellite sequences tend to be highly variable among individuals due to relatively high mutation rates. When the DNA is replicated in meiosis the DNA polymerase enzyme can slip forward or backwards on the repeat units, deleting or adding repeat units to the daughter strand. This means that daughter strands may have slightly fewer or slightly more repeat units than the parent strand.
Geneticists can measure the number of repeat units for a given microsatellite. If several microsatellites are measured on an individual this results in a unique genetic fingerprint.
Microsatellite profiles can be used in many biological applications including:
Each microsatellite gives a small bit of information about an individual but alone cannot identify the individual uniquely. In order to establish unique identity a number of microsatellites must be used on the same tissue and the information from a number of these combined to identify someone at the individual level. For forensic purposes it is possible in principle to exclude two individuals from being a match regardless of how closely they are related by the mismatch of alleles at any locus however it is never possible say two individuals are identical beyond some statistical probability.
For the determination of pedigree information it is possible to match individual offspring to a set of parents assuming genetic information exists for the putative parents and to use this plus other information such as growth performance to determine optimum breeding strategies. This is valuable in cases where physical identification of the parents and offspring are not feasible such as the case with intensive aquaculture. If sufficient numbers of offspring are available even in a mixed group it is possible with a very high degree of certainty to reconstruct parent genotypes solely from the genotype information obtained from the offspring. This can be used to indirectly determine the size of a breeding population in cases where breeding strategies of the species in question is unknown or the parents are not available or readily identifiable.
Comparison of wild populations
For population discrimination rather than looking at individuals allele frequencies, or some other measure of genotypic frequency in one population are compared to the same measures in a second population, again it is a case that rarely a single locus will discriminate clearly between the populations in question but rather they are separated by combining the information from several loci.
In cases where there is either geographic or reproductive isolation of populations it is in some cases possible to visually distinguish two populations as is the case above, the left panel is an inshore population of cod the right panel an offshore population. The dominance of one or two allele types in the left panel shows a clear distinction between the two groups.
Assay of microsatellites
The assay of microsatellite is carried out by polymerase chain reaction (PCR) amplification of a specific microsatellite as defined by the unique primers for the microsatellite. Each microsatellite represents only a tiny portion of the whole genome and as such cannot practically be directly isolated and measured. The use of targeted PCR primers based on the unique sequences around the microsatellite makes it possible to amplify the target microsatellite millions of times and to produce sufficient copies of the microsatellite to easily detect and measure the size of the PCR product and from this determine the number of copies of the repeat present. The amplification of the microsatellite during the PCR reaction also makes it possible to start with very small amounts of genomic DNA be it a hair follicle from a human, a scale from a fish, or needles from a tree. The use of very small amounts of DNA is very important as this allows noninvasive sampling which may be of critical
importance in the case of rare or endangered species or in forensic applications where the available tissue sample may be very small. The assay of a very small portions of the genome also make it possible to study old partially degraded DNA samples which can be recovered from bones or other biological material.
Advantages of PCR-Based Microsatellite Analysis
PCR-based microsatellite analysis has the following advantages over conventional methods of DNA analysis such as Restriction Fragment Length Polymorphism (RFLP):
PCR-based tests are easy to standardize and automate, ensuring reproducible results.
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