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Since Mendel first established the concept of genetic inheritance and the genotype-phenotype connection, scientists have sought to understand and map genetic variation in humans and across a range of other species. There are a number of approaches to genotyping, including analysis of single nucleotide polymorphisms (SNPs), variation of long or short tandem repeats and other DNA fingerprinting methods. Large databases have been amassed to record and share genotyping information and efforts continue to ultimately link variation to the presence of specific traits or disease states. In many instances, however, phenotype and subsequent inheritance is proving to be a more complex story. Genotyping information, however, is still a useful part of the picture and whether this information is gained through PCR, real-time PCR or sequencing methods, Meridian has the right tool to support your research.

Genotyping - Approaches and Methodologies

Characterization of the genetic determinants of specific traits or disease states requires an evaluation of the extant polymorphisms present within a population, family or affected group of individuals.

By far the most informative polymorphisms present within the human genomes are SNPs, occurring in both coding and noncoding regions at a rate of about 1 in 1000 base pairs. Since the Human Genome Project and the subsequent explosion in whole genome sequencing applications, the main SNP database, dbSNP, maintained by the National Center for Biotechnology Information (NCBI) has rapidly accumulated SNP information for over 55 organisms.

A typical SNP genotyping protocol starts with target amplification, using PCR or Real-Time PCR, followed by product detection for allelic discrimination. Mechanisms for allelic discrimination can include the use of restriction enzymes and gel electrophoresis for SNPs that result in restriction fragment length polymorphisms (RFLP). If using Real-Time PCR methods, sequence specific probes can discriminate between different SNPs, or the use of inter-collating dyes followed by melting can produce specific profiles, such as with high resolution melting (HRM) analysis. Pyrosequencing or NGS can also be used to identify, discriminate or catalogue new SNPs within an area of interest. There are hundreds of millions of SNP submissions in the dbSNP database aimed at assisting research and applications in SNP genotyping. Even with the increased use of next generation sequencing (NGS), microarrays and other high throughput technology, with the corresponding surge of data, it is difficult to establish true frequency of many of the reported SNPs within the general population and clear links to disease states or phenotypes are still being attempted for the majority of situations.

There are other ways in which to determine genetic differences that look at variable tandem repeats - two or more DNA base pairs that repeat at specific loci up to hundreds of times and can be sites of high diversity. Microsatellites are a form of short tandem repeat (STR) and are commonly used for linkage mapping and association studies as well as for organism identification. Other approaches to DNA fingerprinting and polymorphism identification include amplified fragment length polymorphism (AFLP) analysis, a way to rapidly generate genetic markers from whole genome analysis without prior knowledge of the genome sequence. Analysis of copy number variation (CNV) is another approach; a form of structural variation, CNV is the result of duplication or deletion events affecting one or more loci, it is widespread in the human genome and often results in functional consequences. From sample preparation through to amplification and detection, Meridian offers a full suite of molecular tools to assist a wide range of genotyping approaches.

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Genotyping – Applications in Human History and Disease

Despite the efficiency of DNA repair mechanisms, variation exists within human populations, with a common SNP (defined as greater than 20% allele frequency in general population) found roughly every 1000 bp.

The lifecycle of a SNP can be thought of in four phases:

New variant appears through random mutation
Variant survives against high odds in early generations (basic probability that a variant will be lost within the first 10 generations is 94%)
Continued survival to substantial frequency through population fluctuations

There is a strong hypothesis that variation between species is an extension of variation within species, regardless, extant SNPs are a product of past mutations and can offer clues about human history. Genotyping or analysis of DNA fingerprints is also used in forensics and paternity testing, looking at specific SNPs or STRs to determine identity or relationship.

Linking genetic differences to phenotype is fairly straight forward for characteristics that follow Mendelian inheritance – one gene influencing one trait. In practice, however, the majority of traits and disease states within human populations are a result of many genes influencing many traits and finding a pattern becomes a complex linkage investigation, mapping alleles to loci and looking for non-random associations connected to the trait of interest. Alternatively, association studies start with a trait of interest and interrogate the genomes of related, non-affected individuals in an attempt to find related differences.

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Genotyping – Other Applications

Genotyping is a valuable tool in a range of other areas outside of human biology. Assessing the genetic variation of common genes has allowed scientists to establish phylogenetic relationships between all living organisms.

Genetic variation databases continue to expand on the number of organisms represented and genotyping continues to be a prominent application

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Bacterial Typing and Resistance Screening

The field of microbiology uses genotyping techniques to regularly classify organisms that cannot easily be grown in culture.

In addition to species detection and identification, sub-typing or strain classification is invaluable for epidemiological studies, for example when studying outbreaks of infectious disease. Genotyping approaches also provide information on the presence of specific characteristics such as virulence or antibiotic resistance and molecular methods can provide rapid information to assist physicians when treating microbial-based disease.

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Plant and Animal Breeding and Agriculture

One of the most fundamental aspects of plant and animal breeding involves the correct selection of individuals with the desired characteristics.

If a genotype/phenotype link is already known, genotyping is an invaluable way to quickly and accurately select appropriate progeny without the need to wait for development of morphological characteristics. Genetic markers for desired traits are continually being investigated and applied in breeding programs as farmers and scientists work together to ensure continued food production for a growing population.

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Conservation Genetics

The understanding that the genetic variability within a population directly impacts the viability of that population is a concept that underscores conservation genetics – a crisis oriented biodiversity management approach.

Genotyping approaches assist in estimating genetic variation within a given population and when combined with breeding characteristics, generation lengths and other variables, risks can be established and appropriate management strategies can be employed to support the continuation of the population in question.

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