Generation of detailed maps using sequence tagged sites

A map designed for a sequencing project to accurately locate the genes and assign the gene function adds direct value to mapping studies. The smallest functional units or genes are present on the chromosome. Hence it is important to study their position and locate them. The recombination frequencies or the number of recombinants found out of the total number of progenies guide in assessing the relative distances between the loci. The mathematical relationship between the map distance and recombination frequencies help to determine map function. Linkage of genes mainly tests whether the genes are present on the same chromosome. Therefore a map is constructed based on the relative positions of the genes. For large genomes, constructing a physical map would add value to the sequencing projects. However, if the purpose is to clone the individual genes, a different approach may be used. The physical mapping techniques do not rely on the presence of alleles to map the genome. The genetic mapping requires the alleles for a given marker. Unavailability of a map leads to an error-prone assembly of the genome sequence. The genetic maps have a poor resolution and inaccuracy. These properties are refined using a physical map.

Image 1: STS Mapping (Cloned DNA fragments showing markers)

Molecular biology techniques are used to construct a genome map. The plethora of physical mapping involves restriction mapping, FISH, and STS mapping. Restriction sites in small DNA molecules are possible in restriction mapping with a limitation in eukaryotic chromosomes. FISH is a good choice for mapping genomes. However, it takes a lot of time for mapping large genomes. More specific approach for mapping large genomes is required. Sequence tagged sites (STS) guide very accurately in physical mapping. This type of mapping is known as STS mapping. A sequence tagged site though seems complicated by its name, is not so complicated. In fact, it is a very specific approach. It is any site in the chromosome that is identified by a known unique DNA sequence. Mapping large genomes require a high resolution and rapid technique with less demand in technicality.

The limitations of FISH being difficult to conduct and accumulate the data in a single experiment, STS mapping may meet the requirement of providing map positions more than three markers in one go. A short DNA sequence with 100-500 base pairs is easy to recognize because it occurs only once in the chromosome or a genome. Such a site or a sequence is useful for physical mapping. STS mapping requires such a collection of overlapping fragments from the genome. The entire chromosome is used to obtain the mapping reagent or a collection of overlapping fragments. It helps to identify the marker position.

Properties of Sequence Tagged Sites:

As discussed earlier, sequence tagged sites are small DNA sequences that are unique. Two main properties determine sequence tagged sites. The DNA sequence must be known. Hence, a PCR assay can be set up. A PCR assay of known DNA sequence determines the STS on different fragments. The DNA fragment must have a unique location on a chromosome. If the STS are positioned more than once in the genome, it becomes difficult to map. Mainly repetitive DNA has high chances of having more than once positioned sequences. Thus STS mapping does not include repetitive DNA sequences. The reason for using PCR instead of hybridization lies in its automatic mechanisms and efficiency. It is difficult to include those fragments whose sequence is not known to us. Therefore the foremost criteria for a genome mapping are to know a sequence. The probability that the two closely linked markers determined by the fragments found on the same chromosome.

Distantly linked markers may be present in different fragments. A collection of fragments involves many fragments with closely linked or distantly linked markers. The frequency at which breaks occur between the two markers decides the map distance.

Image 2: Fragment collection (STS mapping)

Sources of sequence tagged sites (STS):

Three main sources of sequence tagged sites include expressed sequence tags (EST), SSLPs, and random genomic sequences.

·        Expressed sequence tags (ESTs): The cDNA clones are analyzed to obtain short sequences known as expressed sequence tags or ESTs. The sequence derived from the cDNA library is unique. A sequence transcribed in some tissue or at some stage of the developmental process is used to derive a unique sequence. Thus, an EST mapped through a specific mapping procedure identifies a unique gene locus. EST markers are produced using PCR. It involves oligonucleotide primers based on cDNA sequence. They correspond to protein-coding genes. So, the unique ESTs are capable of becoming STS.

·        SSLPs: Simple sequence length polymorphisms are arrays of repeat sequences displaying length variations. SSLPs contain alleles with a different number of repeat units that are multi-allelic. Two main types of SSLPs include minisatellites and microsatellites. Polymorphic SSLPs are usually preferred.

·        Random genomic sequences: They are randomly cloned sequences. Randomly spanning the available online databases help to obtain random genomic sequences. However, they are known sequences.

Fragment collection for STS mapping:

The collection of DNA fragments are known as a mapping reagent. The fragments are present in the entire chromosome. Each point has an average of five fragments. The markers may be near or far on the fragments. Mapping reagents assemble in two ways such as clone library and radiation hybrids. Rodent cell lines support the radiation mapping techniques. Different fragments of the second genome consist of the rodent cell line. Irradiation techniques are used to construct these cell lines. Hence they are mapping reagents in studying large genomes. A clone library is another mapping reagent. It is a collection of clones representing an entire genome. These clone collections supply individual clones of interest. Let us know the two mapping reagents in detail.

Radiation hybrids:

The human chromosomes paved way in the development of radiation hybrids. During the early 1970’s, experimenters exposed human cells to 3000-8000 rad doses of X-rays, leading to chromosome breakage. However, this treatment was lethal for human cells. The irradiated cells propagated on fusion with non-irradiated hamster cells. Polyethylene glycol or Sendai viral exposure led to the fusion of both the cells. However, all hamster cells are not capable of accepting human chromosomes. The hamster cells, therefore, need to undergo a selection process. Some hamster cells are unable to make thymidine kinase or hypoxanthine phosphoribosyl transferase. So the cells are grown in a medium containing hypoxanthine, aminopterin and thymidine medium (HAT). The fused cells are cultured in the HAT medium.

Hybrid hamster cells grow on this medium. It indicates that these cells have accepted human chromosomes. The hybrid cells consist of human DNA inserted into hamster chromosomes. These fragments are 5-10 Mb in size. The collection of hybrids is known as radiation hybrid panels. It is a mapping reagent. The rodent cells may also be used to obtain radiation hybrid panels. The rodent cell lines consist of human DNA fragments in the rodent nucleus. Sometimes the hybrid rodent cells are fused with hamster cells. Such hybrids may contain both human and mouse chromosomes or a mixture of both. Specific probes help to identify hybrid cells containing human DNA. The probes are specific sequences identifying human DNA. Examples include SINEs or short interspersed nuclear element called as Alu. The Alu elements have a copy number of over a million. Two types of radiation hybrid panels are known so far. They include single chromosome panels and whole genome panels. Let us know the difference between the two.

Single chromosome panel	Whole genome panel
Only a few hybrids are required	A few hundred hybrids are required.
PCR screening involved convenience in handling	Involved less convenience in handling
Involved irradiation of mouse cell containing more mice DNA and less human DNA.	Irradiation of human DNA
Human DNA hybrid is less in content	Higher human DNA hybrid content
The human genome project avoided the approach due to less human DNA hybrid content.	The human genome project utilized the approach due to high human DNA hybrid content.

 Table: Difference between single chromosome panel and the whole genome panel

Clone library:

A collection of clones represents the human genome. They are used to obtain individual clones of interest. A clone library is obtained by breaking the genome into fragments and thereby cloning them into the vector. Hence, a clone library consisting of large genome fragments is a mapping reagent. A clone library is a chromosome specific library. It is possible to separate chromosomes using a clone library. They have sufficient information for STS mapping. The STS analysis determines the clones consisting of overlapping DNA fragments enabling clone contigs to build-up.

References:
[1] Molecular Biology, David P. Clark, Nanette J. Pazdernik
[2] Human Molecular Genetics 3, Volume 3, T. Strachan, Andrew P. Read

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