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Genetic Marker

Submitted By:

Purnima

Bioinformatics Deptt.

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Definition

  • A genetic marker is a DNA sequence with a known physical location on a chromosome.
  • It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be observed.
  • A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like minisatellites.

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Genetic Marker

  • Genetic markers can help link an inherited disease with the responsible gene. DNA segments close to each other on a chromosome tend to be inherited together.
  • Genetic markers are used to track the inheritance of a nearby gene that has not yet been identified, but whose approximate location is known. The genetic marker itself may be a part of a gene or may have no known function.

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Types of Marker

Some commonly used types of genetic markers are:

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RFLP

  • Restriction fragment length polymorphism (RFLP) is a technique invented in 1984 by the English scientist Alec Jeffreys during research into hereditary diseases. It is used for the analysis of unique patterns in DNA fragments in order to genetically differentiate between organisms – these patterns are called Variable Number of Tandem Repeats (VNTRs).
  • Restriction fragment length polymorphism (abbreviated RFLP) refers to differences (or variations) among people in their DNA sequences at sites recognized by restriction enzymes.
  • Such variation results in different sized (or length) DNA fragments produced by digesting the DNA with a restriction enzyme.
  • RFLPs can be used as genetic markers, which are often used to follow the inheritance of DNA through families.

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RFLP

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PRINCIPLE OF RFLP

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  • Restriction endonucleases are enzymes that cut lengthy DNA into short pieces. Each restriction endonuclease targets different nucleotide sequences in a DNA strand and therefore cuts at different sites.
  • The distance between the cleavage sites of a certain restriction endonuclease differs between individuals. Hence, the length of the DNA fragments produced by a restriction endonuclease will differ across both individual organisms and species.

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RFLP analysis�

  • The basic technique for the detection of RFLPs involves fragmenting a sample of DNA with the application of a restriction enzyme, which can selectively cleave a DNA molecule wherever a short, specific sequence is recognized in a process known as a restriction digest.
  • The DNA fragments produced by the digest are then separated by length through a process known as agarose gel electrophoresis and transferred to a membrane via the Southern blot procedure. 
  • Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe.
  • A restriction fragment length polymorphism is said to occur when the length of a detected fragment varies between individuals, indicating non-identical sequence homologies.
  • Each fragment length is considered an allele, whether it actually contains a coding region or not, and can be used in subsequent genetic analysis.

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Applications

RFLP has been used for several genetic analysis applications since its invention.

Some of these key applications of RFLP are listed below:

  • To determine the status of genetic diseases such as Cystic Fibrosis in an individual.
  • To determine or confirm the source of a DNA sample such as in paternity tests or criminal investigations.
  • In genetic mapping to determine recombination rates that show the genetic distance between the loci.
  • To identify a carrier of a disease-causing mutation in a family.

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RAPD

  • RAPDs are DNA fragments amplified by PCR using short synthetic primers (generally 10 bp) of random sequence. These oligonucleotides serve as both forward and reverse primer, and are usually able to amplify fragments from 1-10 genomic sites simultaneously. Amplified fragments, usually within the 0.5-5 kb size range, are separated by agarose gel electrophoresis, and polymorphisms are detected, after ethidium bromide staining, as the presence or absence of bands of particular sizes. These polymorphisms are considered to be primarily due to variation in the primer annealing sites, but they can also be generated by length differences in the amplified sequence between primer annealing sites.

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Strengths�

  • The main advantage of RAPDs is that they are quick and easy to assay. Because PCR is involved, only low quantities of template DNA are required. Since random primers are commercially available, no sequence data for primer construction are needed. Moreover, RAPDs have a very high genomic abundance and are randomly distributed throughout the genome.

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Weaknesses�

  • The main drawback of RAPDs is their low reproducibility, and hence highly standardized experimental procedures are needed because of their sensitivity to the reaction conditions.
  • RAPD analyses generally require purified, high molecular weight DNA, and precautions are needed to avoid contamination of DNA samples because short random primers are used that are able to amplify DNA fragments in a variety of organisms.
  • Altogether, the inherent problems of reproducibility make RAPDs unsuitable markers for transference or comparison of results among research teams working in a similar species and subject.
  • As for most other multilocus techniques, RAPD markers are not locus-specific, band profiles cannot be interpreted in terms of loci and alleles (dominance of markers), and similar sized fragments may not be homologous.

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Applications�

  • RAPDs have been used for many purposes, ranging from studies at the individual level (e.g. genetic identity) to studies involving closely related species.
  • RAPDs have also been applied in gene mapping studies to fill gaps not covered by other markers.
  • Variants of the RAPD technique include Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) which uses longer arbitrary primers than RAPDs, and DNA Amplification Fingerprinting (DAF) that uses shorter, 5-8 bp primers to generate a larger number of fragments. Multiple Arbitrary Amplicon Profiling (MAAP) is the collective term for techniques using single arbitrary primers.

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SNPs

  • Single nucleotide polymorphisms, frequently called SNPs (pronounced “snips”), are the most common type of genetic variation among people. Each SNP represents a difference in a single DNA building block, called a nucleotide. For example, a SNP may replace the nucleotide cytosine (C) with the nucleotide thymine (T) in a certain stretch of DNA.
  • SNPs occur normally throughout a person’s DNA. They occur almost once in every 1,000 nucleotides on average, which means there are roughly 4 to 5 million SNPs in a person's genome. These variations occur in many individuals; to be classified as a SNP, a variant is found in at least 1 percent of the population. Scientists have found more than 600 million SNPs in populations around the world.

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SNPs

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SNPs

  • Single nucleotide polymorphism, or SNP. If you are reading a news story where it says, for example, scientists find the genetic contributors to diabetes or some other condition or trait, you're probably reading about SNPS. A SNP is a one-letter place where your genome varies from another genome sequence. Thanks to the Human Genome Project, we have found that these single letter changes in our genetic code are placed all across our genomes.
  • We can see that the patterns vary between people and even between populations. If we want to identify genetic contributors to a common complex disease like diabetes, we can group together thousands of people who have diabetes and compare their SNP patterns to thousands of people who do not have diabetes. With enough people in our study, we can use the SNPs as markers to see that certain areas of the genome appear to be the same in people who have diabetes, and that tells us where we should look in more detail for a genetic cause.

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Types�

  • Single-nucleotide polymorphisms may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions (regions between genes). SNPs within a coding sequence do not necessarily change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code.
  • SNPs in the coding region are of two types: synonymous SNPs and nonsynonymous SNPs. Synonymous SNPs do not affect the protein sequence, while nonsynonymous SNPs change the amino acid sequence of protein.
  • SNPs in non-coding regions can manifest in a higher risk of cancer, and may affect mRNA structure and disease susceptibility. Non-coding SNPs can also alter the level of expression of a gene, as an eQTL (expression quantitative trait locus).

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Frequency�

  • Within a genome

The genomic distribution of SNPs is not homogenous; SNPs occur in non-coding regions more frequently than in coding regions or, in general, where natural selection is acting and "fixing" the allele (eliminating other variants) of the SNP that constitutes the most favorable genetic adaptation.[18] Other factors, like genetic recombination and mutation rate, can also determine SNP density.

  • Within a population

There are variations between human populations, so a SNP allele that is common in one geographical or ethnic group may be much rarer in another. However, this pattern of variation is relatively rare; in a global sample of 67.3 million SNPs, the Human Genome Diversity Project "found no such private variants that are fixed in a given continent or major region. The highest frequencies are reached by a few tens of variants present at >70% (and a few thousands at >50%) in Africa, the Americas, and Oceania. By contrast, the highest frequency variants private to Europe, East Asia, the Middle East, or Central and South Asia reach just 10 to 30%."

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Applications�

  • Association studies can determine whether a genetic variant is associated with a disease or trait.
  • A tag SNP is a representative single-nucleotide polymorphism in a region of the genome with high linkage disequilibrium (the non-random association of alleles at two or more loci). Tag SNPs are useful in whole-genome SNP association studies, in which hundreds of thousands of SNPs across the entire genome are genotyped.
  • Haplotype mapping: sets of alleles or DNA sequences can be clustered so that a single SNP can identify many linked SNPs.
  • Linkage disequilibrium (LD), a term used in population genetics, indicates non-random association of alleles at two or more loci, not necessarily on the same chromosome. It refers to the phenomenon that SNP allele or DNA sequence that are close together in the genome tend to be inherited together. LD can be affected by two parameters (among other factors, such as population stratification): 1) The distance between the SNPs [the larger the distance, the lower the LD]. 2) Recombination rate [the lower the recombination rate, the higher the LD].
  • In genetic epidemiology SNPs are used to estimate transmission clusters.

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Sequence-Tagged Sites (STS)�

Sequence-Tagged Site (STS)is a relatively short, easily PCR-amplified sequence (200 to 500 bp) which can be specifically amplified by PCR and detected in the presence of all other genomic sequences and whose location in the genome is mapped.

  • The STS concept was introduced by Olson et al (1989). In assessing the likely impact of the Polymerase Chain Reaction (PCR) on human genome research, they recognized that single-copy DNA sequences of known map location could serve as markers for genetic and physical mapping of genes along the chromosome. The advantage of STSs over other mapping landmarks is that the means of testing for the presence of a particular STS can be completely described as information in a database: anyone who wishes to make copies of the marker would simply look up the STS in the database, synthesize the specified primers, and run the PCR under specified conditions to amplify the STS from genomic DNA.

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STS continue……

  • The uniqueness of the sequence is established by demonstrating that it can be uniquely amplified by the PCR. The DNA sequence of an STS may contain repetitive elements, sequences that appear elsewhere in the genome, but as long as the sequences at both ends of the site are unique, unique DNA primers complementary to those ends can be synthesized, the region amplified using PCR, and the specificity of the reaction demonstrated by gel electrophoresis of the amplified product.

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Applications of STS�

  • STSs are very helpful for detecting microdeletions in some genes. For example, some STSs can be used in screening by PCR to detect microdeletions in Azoospermia (AZF) genes in infertile men.
  • Identification of genes in elephants could provide additional information for evolutionary studies and for evaluating genetic diversity in existing elephant populations. Sequence tagged sites (STSs) were identified in the Asian and the African elephant for the following genes: melatonin receptor 1a (MTNR1A), retinoic acid receptor beta (RARB), and leptin receptor (LEPR) (Burk et al., 1998).

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References

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