SSCP

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).

 

Advantages

SSCP screening has two primary advantages as a mutation-screening technique:

  • You can screen for mutations in a specified DNA region by choosing PCR primers that span that region.
  • You can screen a large number of samples because the technique is simple and fast.

The only step necessary after PCR amplification is a heat denaturation in formamide and NaOH.

 

Limitations

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:

  • Multiple gel running conditions
    • temperature (~ room temperature, ~ 4oC)
    • addition of additives e.g. glycerol (5-10%); sucrose (10%)
  • Different gel matrices
    • Low polyacrylamide cross linker ratios (49:1 acrylamide: bis-acrylamide) (5-10% gels)
    • MDE, GeneAmp, Hydrolink gels
    • Agarose gels (resolution of minisatellite isoalleles (same length , but a few base pair substitutions within the minisatellite repeat) as long as 6.3kb; Monkton and Jeffreys, 1995).
  • High throughput SSCP: use of sharkstooth combs, multichannel pipettes, microtitre plates, staggered repetitive loadings on two gels poured between three plates resulted in 1000 PCR reactions prepared and analysed in 24 man hours using five 40cm x 30cm gel tanks. Used for the analysis of exon 3 of the LDLR in patients with familial hypercholesterolaemia (Whittall et al, 1995).
  • rSSCP: RNA-SSCP: single stranded RNA should have a larger repertoire of secondary structure, since RNA basepairing is more stable than RNA-DNA basepairing, and might be expected to adopt more conformational structure and hence be more sensitive to sequence changes. Efficiency of detection: 70% for rSSCP in comparison to 35% for SSCP (for 2.6kb of Factor IX gene). However, it is more inconvenient to make RNA strands (involving introducing RNA polymerase promoters etc.) and although the abundance of transcript allows for ethidium bromide detection of the rSSCPs, the added complexity and expense has precluded widespread use (Sarkar et al, 1992a).

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).

 

 

Microsatellites

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.

 

Background

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.
If allele frequencies are known, highly polymorphic microsatellite loci are very useful for identifying individuals in a population and for determining the probability that two individuals are related. In addition, the even distribution of microsatellites in the genome makes them good markers for constructing genetic maps.

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:

  • To identify unique individuals in a population: people, animals or plants
  • In forensic science, to compare suspects to crime scene stains
  • To determine the paternity or maternity of individuals
  • To estimate population size for wild populations
  • To determine if populations have suffered genetic bottlenecks
  • For phylogenetic studies in applications involving very closely related subspecies
  • For genetic mapping
  • To determine if there is population subdivision (ie local inbreeding) in wild populations

Forensic applications

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.

Pedigree determination

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):

  • The small size of microsatellite loci improves the chance of obtaining a result, particularly for samples containing minute amounts of DNA and/or degraded DNA.
  • The small size range of microsatellite loci makes them ideal candidates for co-amplification while keeping all amplified alleles smaller than 350 base pairs. Many microsatellite loci can therefore be typed from a single PCR.
  • Microsatellite alleles have discrete sizes, allowing for simplified interpretation of results.
  • PCR-based tests are rapid, giving results in 24 hours or less.

PCR-based tests are easy to standardize and automate, ensuring reproducible results.

References:

Youil R, Kemper BW, Cotton RG (1995) Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII. Proc Natl Acad Sci USA 92(1): 87-91.

Youil R, Kemper BW, Cotton RG (1996) Detection of 81 of 81 known mouse beta-globin promoter mutations with T4 endonuclease VII-the EMC method. Genomics 32 (3): 431-435.

 

Hayashi K and Yandell DW. (1993) How sensitive is PCR-SSCP? Hum Mut 2: 338-346.

Iwahana H, Fujimura M, Takahashi Y, Iwabuchi T, Yoshimoto K Itakura M. (1996) Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labelling of PCR products. Biotechniques 21(3): 510-514.

Katsuragi K, Kitafishi K, Chilba W, Ikeda S, Kinoshita M. (1996) Fluorescence-based PCR-SSCP analysis of p53 gene by capillary electrophoresis. J Chromatogr A 722(1-2): 311-320.

Lancaster JM, Berchuck A, Futreal PA, Wiseman RW. (1997) Dideoxy fingerprinting assay for BRCA1 mutation analysis. Mol Carcinog 19(3): 176-179.

Liu Q and Sommer SS. (1994) Parameters affecting the sensitivities of dideoxy fingerprinting and SSCP PCR Methods Appl 4(2): 97-108.

Liu Q and Sommer SS. (1995) Restriction endonuclease fingerprinting (REF): as sensitive method for screening mutations in long contiguous segments of DNA. Biotechniques 18(3): 470-477.

Liu Q, Feng J, Sommer SS. (1996) Bi-directional dideoxy fingerprinting (Bi-ddF): a rapid method for quantitative detection of mutation in genomic regions of 300-600 bp. Hum Mol Gen 5(1): 107-114.

Lo Y, Patel P, Mehal WZ, Fleming KA, Bell JI, Wainscoat JS. (1992) Analysis of complex genetic systems by ARMS-SSCP: application to HLA genotyping. Nucleic Acids Res 20(5): 1005-1009.

Makino R, Yazyu H, Kishimoto Y, Sekiya T, Hayashi K (1992) F-SSCP: fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. PCR Methods Appl 2(1):10-13.

Martincic D and Whitlock JA (1996) Improved detection of p53 point mutations by dideoxyfingerprinting (ddF). Oncogene 13(9): 2039-2044.

Monkton DG and Jeffreys A. (1994) Minisatellite isoalleles can be distinguished by SSCP analysis in agarose gels. Nucl Acids Res 22: 2155-2157.

Orita M, Suzuki Y, Sekiya T, Hayashi K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874-879.

Sarkar G, Yoon HS, Sommer SS. (1992a) Screening for mutations by RNA single-stranded conformation polymorphism (rSSCP): comparison with DNA-SSCP. Nucleic Acids Res 20(4): 871-878.

Sarkar G, Yoon HS, Sommer SS. (1992b) Dideoxy fingerprinting (ddF); a rapid and efficient screen for the presence of mutations. Genomics 13(2): 441-443.

Whittall R, Gudnason V, Weavind GP, Day LB, Humphries SE, Day INM. (1995) Utilities for high throughput use of the SSCP method: screening of 791 patients with familial FH for mutation in exon 3 of the LDLR gene. J Med Genet 32: 509-515.

 

 

 

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