Genotyping

Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents. Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual.

Nonhuman Animals

Determining Sex

Birds

In avian species where external sexual dimorphism is absent or subtle, such as monomorphic species in captivity and juveniles in the wild, sexing birds for research purposes can utilize genetics. DNA samples be collected from feathers and the blood of birds. Birds possess a ZW sex determination system, in which females are heterogametic (ZW) and males are homogametic (ZZ). This is in contrast to the XY sex determination system of humans where males are heterogametic (XY) and females are homogametic (XX).

A widely used genetic marker for avian sexing is the CHD1 gene, which exists in slightly different forms on the Z and W chromosomes, called CHD1Z and CHD1W, respectively. These gene variants differ in the number of base pairs, enabling their detection through amplification by Polymerase Chain Reaction (PCR) followed by separation by gel electrophoresis.

CHD1 Primers for Genotyping

There are many well-developed and validated primers that amplify a certain region of the CHD1 gene that shows a difference in size between the W and Z chromosome variants. Five sets of two primers for the CHD1 gene, each (166F/279R, 1237L/1272H, 2550F/2718R, P8/P2, P3/P2) have been tested to show different lengths of products in a wide range of roughly 80 bird species ranging from songbirds to chicken. These sets of primers contain one primer for each of the sex chromosomes. When amplified PCR products are separated via gel electrophoresis, males (ZZ) display a single band (two identical CHD1Z genes), while females (ZW) exhibit two distinct bands corresponding to both gene variants (CHD1Z and CHD1W). Sex can then be determined by identifying the number of bands for each bird being genotyped.

Limitations to CHD1 Genotyping

The CHD1 molecular sexing assay can be used in a wide range of applications, from conservation biology to sexing avian models of behaviour. PCR-based sex determination is of use when morphological indicators are absent or unavailable. Despite its' ease of use and convenience, there are some limitations with using CHD1 as the main marker for determining sex. Because the nucleotide length difference between the CHD1W and CHD1Z gene varies between species, difficulties with genotypic sexing using the P2/P8 and 1237L/1272H CHD1 primers have been reported. As a result, alternative primers and markers have been provided to obtain more reliable genotyping results. These methods utilize different post-PCR modifications, and protocols. These methods include Single Strand Conformation Polymorphism and Restriction Fragment Length Polymorphism that further processes CHD1 PCR products.

Applications

Microbial

Genotyping applies to a broad range of individuals, including microorganisms. For example, viruses and bacteria can be genotyped. Genotyping in this context may help in controlling the spreading of pathogens, by tracing the origin of outbreaks. This area is often referred to as molecular epidemiology or forensic microbiology.

Human

Humans can also be genotyped. For example, when testing fatherhood or motherhood, scientists typically only need to examine 10 or 20 genomic regions (like single-nucleotide polymorphism (SNPs)), which represent a tiny fraction of the human genome.

When genotyping transgenic organisms, a single genomic region may be all that needs to be examined to determine the genotype. A single PCR assay is typically enough to genotype a transgenic mouse; the mouse is the mammalian model of choice for much of medical research today.

Tuberculosis

Genotyping is used in the medical field to identify and control the spread of tuberculosis (TB). Originally, genotyping was only used to confirm outbreaks of tuberculosis; but with the evolution of genotyping technology it is now able to do far more. Advances in genotyping technology led to the realization that many cases of tuberculosis, including infected individuals living in the same household, were not actually linked. This caused the formation of universal genotyping in an attempt to understand transmission dynamics. Universal genotyping revealed complex transmission dynamics based on things like socio-epidemiological factors. This led to the use of polymerase chain reactions (PCR) which allowed for faster detection of tuberculosis. This rapid detection method is used to prevent TB. The addition of whole genome sequencing (WGS) allowed for identification of strains of TB which could then be put in a chronological cluster map. These cluster maps show the origin of cases and the time in which those cases arose. This gives a much clearer picture of transmission dynamics and allows for better control and prevention of transmission. All of these different forms of genotyping are used together to detect TB, prevent its spread and trace the origin of infections. This has helped to reduce the number of TB cases.

Agricultural

Many types of genotyping are used in agriculture. One type that is used is genotyping by sequencing because it aids agriculture with crop breeding. For this purpose, single nucleotide polymorphisms (SNPs) are used as markers and RNA sequencing is used to look at gene expression in crops. The knowledge gained from this type of genotyping allows for selective breeding of crops in ways which benefit agriculture. In the case of alfalfa, the cell wall was improved through selective breeding that was made possible by this type of genotyping. These techniques have also resulted in the discovery of genes that provide resistance to diseases. A gene called Yr15 was discovered in wheat, which protects against a disease called yellow wheat rust. Selective breeding for the Yr15 gene then prevented yellow wheat rust, benefiting agriculture.

Techniques

Restriction Fragment Length Polymorphisms

A restriction fragment length polymorphism (RFLP) is a variation between different people at sites of the genome recognized by restriction enzymes. DNA containing different restriction sites will be cut by bacterial restriction enzymes differently and this can be seen using gel electrophoresis. When running the sample through, a successfully cleaved sample will contain two bands, while the sample with a different restriction site polymorphism will have one band as it had not been cleaved. A small change is enough to cause that restriction site to deny the restriction enzyme. This method is often used to trace the inheritance of DNA through families.

Random Amplified Polymorphic Detection

The random amplified polymorphic detection (RAPD) method relies on PCR methods to amplify and isolate lengths of DNA fragments. Oligonucleotide primers are used which bind to denatured DNA fragments which have been produced through heat treatment. Two primers, one to define the starting point and ending point of PCR DNA synthesis, are used in this process. The fragments of DNA will range from two to three kbp and different primers are tried until the desired trait is isolated from the genome. This method is useful in locating small differences to differentiate between species.

Amplified Fragment Length Polymorphisms

The amplified fragment length polymorphism (AFLP) detection method is much like RAPD as it also relies on PCR amplification of DNA, with the difference being that this process is more precise but also more time consuming than the RAPD counterpart. It also does not require random primers, instead the DNA is digested by restriction enzymes and the ends are then ligated to adaptors which allow for specification of strands when performing PCR amplification, this is where the improved precision of this method comes from.

DNA Microarrays/Beads

This process uses specific oligonucleotides which are placed on a DNA microarray which bind to complementary strands of DNA. This method is optimal for detecting single nucleotide polymorphisms in the DNA. The DNA will bind to the oligonucleotide bead up until one base pair before the SNP, where a single labeled nucleotide will be incorporated. This will be seen through dyes and fluorescently labeled proteins which indicate which SNP can be found at the locus of interest.

Ethical concerns

The ethics of genotyping humans have been a topic of discussion. The rise of genotyping technologies will make it possible to screen large populations of people for genetic diseases and predispositions for disease. The benefits of population wide genotyping have been contended by ethical concerns on consent and general benefit of wide span screening.

Psychological

Genotyping identifies mutations that increase susceptibility of a person to develop a disease, but disease development is not guaranteed in most cases, which can cause psychological damage.

Discrimination

Discrimination can arise from various genetic markers identified by genotyping, such as athletic advantages or disadvantages in professional sports or risk of disease development later in life.

Availability

Much of the ethical concerns surrounding genotyping arise from information availability, as in who can access the genotype of an individual in various contexts.

See also

• Mendelian error – Error in the mendelian inheritance
• Quantitative trait locus – DNA locus associated with variation in a quantitative trait
• SNP genotyping – Measurement of genetic variations

External links

• International HapMap Project Archived 2014-04-16 at the Wayback Machine
• resources for genotyping microorganisms