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.
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 polymerase chain reaction (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 kilo base pairs 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 (SNPs) 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.
Genome Wide Association Studies This is a method which takes the sequenced genome and compares different genomes to find genomic variants correlated with different traits or diseases. In this method, thousands of SNPs are studied and compared in sample sizes ranging up to millions of genomes. If certain SNPs are found to be statistically significant over the sample, the genes are identified which contain those SNPs and that gene is then correlated with the trait of interest. This method can be useful implications in the future for personalized medicine.
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 SNPs).
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.
Ancient DNA Studies Ancient DNA (aDNA) studies have been very important in understanding human evolution, but the samples are often highly degraded. This means there are certain steps and techniques required to ensure accuracy and readability of ancient genomes. To begin the researchers must ensure the DNA being studied is not contaminated with any recent DNA and only contains aDNA. To do this researchers will often look for terminal deamination as this is typically seen in aDNA. They will also often compare the sample with moderns human DNA to see if it is similar. Since aDNA can be sparse, certain techniques involve a targeted enrichment of the area of interest in the aDNA which can increase the resolution of these regions.
They also must work around the various DNA changes that can occur after the death of the individual. One very common form of damage is the deamination of cytosines. This causes the genome to be misread with C to T and G to A mistakes. This is often mitigated by treating the DNA with a USER reagent. This is a mix of uracil-DNA glycosylase and endonuclease VIII. This functions by removing the uracil and cleaving the site which it was removed from.
Pseudohaploid Genotyping This genotyping method is often used for aDNA studies when the DNA reads available are low-coverage or when there is a large number of low coverage genomes being studied. This method does not determine the true diploid genotype but instead researchers will specify which SNPs they would like to focus on and read from the genome. Tools that allow this method include programs like pileupCaller and bam-caller.
Probabilistic Genotyping Probabilistic genotyping is best used when the aDNA available is of high quality. This method finds the probability that the ancient genotype was present in the aDNA, often using modern DNA genotypes as reference samples. Programs used for this type of genotyping include snpAD, ATLAS, bcftools, GATK, or ANGSD.
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 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, 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.
Sex Determination in Nonhuman Animals
Determining Sex in Birds In avian species where external phenotypic sexual dimorphism is absent or subtle, such as monomorphic species in captivity and juveniles in the wild, sexing birds for research purposes can utilize molecular genetic methods. DNA samples be collected from feathers and 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 PCR followed by gel electrophoresis separation.
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 PCR 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 bands corresponding to each gene variant of different sizes (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 between species. These methods utilize different post-PCR modifications, and protocols, including Single Strand Conformation Polymorphism and Restriction Fragment Length Polymorphism that further processes CHD1 PCR products.
Determining Sex in Chinese soft-shelled turtles (Pelodiscus sinensis)