The below mentioned article provides notes on genomes: 1. Introduction to Genomics 2. Mapping Approach for Sequencing Human Genome 3. Meiotic Recombination and Cytogenetic Maps 4. Radiation Hybrid Mapping 5. Physical Mapping of the Genome 6. Sequencing the Genome 7. Optical Mapping of Whole Genome 8. Synthetic Genomics.
Introduction to Genomics:
Recombinant DNA technology has been extended to studies of whole genomes by mapping and sequencing techniques. Genomics is, therefore, the molecular mapping and characterisation of whole genomes and their gene products.
The small genome of the bacteriophage φX174 was the first one to be sequenced, followed by plasmid and viral genomes. Subsequently genomes of eukaryotes such as yeast, Drosophila, rice plant and chimpanzee were sequenced. The complete sequence of the circular genome in the human mitochondrion (16159 bp) was determined in 1981.
The most recent achievement is the successful completion of the human genome project (HGP) in 2003. The project had aimed to identify all of the approximately 20,000 to 25,000 genes in human DNA; determine the sequences of the three billion base pairs that comprise the human genome; information retrieved from the project to be stored in databases; develop improved tools for analysis of data; transfer related technologies to the private sector; and to address the ethical, legal and social issues (ELSI) pertaining to this project.
Among the special experimental techniques that were devised include those for handling and sequencing millions of clones. Hi-tech automation for decoding information and computer-driven robotics were employed.
Bioinformatics played a central role in data analysis. The project was coordinated jointly by the U.S. Department of Energy (DOE) and National Institutes of Health (NIH) involving cooperative efforts of many scientists and engineers from several countries. Though HGP is finished, analysis of data will continue for many years.
Functional genomics refers to analysis of the function of genes through expression of gene products, as well as the non-gene sequences in the entire genome. It includes study of the control of gene regulation, interactions, regulation of RNAs and proteins. Gene expression can be analysed through study of the all the expressed mRNA transcripts, known as the transcriptome, and through polypeptides called the proteome.
The earlier attempts in making genetic maps were based on recombination frequencies. Using suitable organisms, such as yeast and Drosophila, large-scale crosses were made between different mutant strains. The methodology relied on the concept that the further apart in map distance, two loci are on a chromosome, greater is the chance that a crossover will occur between them during meiosis.
Recombinants in the offsprings resulting from crossovers were scored and map distances were recorded. Thus low resolution chromosomal maps of genes producing known mutant phenotypes were constructed.
This approach cannot be used for mapping the human genome because of its large size (3000 Mb or 3 billion base pairs), and inability to set up large-scale matings between people carrying different inherited diseases.
Instead, human genetic mapping can be carried out through analysis of DNA sequence polymorphisms in the population, that are naturally occurring DNA sequence differences which do not produce visible phenotypes. We describe here the various approaches and technologies which have resulted in mapping the human genome.
Whole genome mapping is approached in two ways: by mapping or shotgun cloning. Mapping the whole genome involves the development of high resolution genetic and physical maps in order to generate DNA segments of increasing resolution, and then to determine the sequence of the fragments. The genetic maps can be based on the order of markers by meiotic recombination, or by co-localisation of genes in individual fragments of chromosomes.
Physical maps provide a view of how the clones from genomic clone libraries are distributed throughout the genome. The other approach uses whole genome shotgun cloning in which the genome is broken up into random overlapping fragments. Then to sequence the fragments and assemble the segments using computational methods.
The Mapping Approach for Sequencing Human Genome:
The initial goal of preparing a genetic and physical map is to obtain closely spaced markers throughout the genome. The Human Genome Project (HGP) started out with the aim of constructing a high density genetic map with at least one genetic marker per one Mb (about one map unit) of the genome.
The procedure starts by developing low resolution genetic maps using recombinational mapping of inherited differences, or cytogenetic mapping methods. Prepare physical maps indicating positions of individual cloned DNA fragments of each chromosome. These genetic and physical maps are then integrated with molecular maps at higher resolution. A dense array of markers is obtained that can be used directly in gene cloning.
Thereafter, conduct large scale genomic DNA sequence analysis to produce a complete sequence map of each chromosome. The idea of progressively increasing resolution of analysis is a central feature in mapping the genome.
Meiotic Recombination and Cytogenetic Maps:
Meiotic linkage mapping is carried out in organisms such as yeasts, fungi (tetrad analysis), Drosophila and some plants. These methods are not usable in case of humans owing to lack of information from crosses; small size of progeny does not allow accurate determination of linkage; and very large size of human genome with 24 linkage groups (22 autosomal and 2 sex chromosomes).
In humans pedigree analysis is one of the approaches used to determine linkage relationships between human genes, including pedigree analysis for determining recombination frequency, as well as molecular methods.
Cytogenetic Maps:
High resolution cytogenetic maps can be developed by determining locations of DNA markers in relation to visible chromosome bands, puffs and positions of centromeres. One of the methods known as in situ hybridisation was developed in 1970 by Pardue and Gall for locating repeated DNA sequences in eukaryotic chromosomes.
In this technique chromosomes are spread on a glass slide and denatured by appropriate treatment. A cloned DNA sequence can be used to make a labeled probe for hybridisation to chromosomes. A labelled denatured probe will hybridise to homologous sequences in single strands of chromosomes in situ.
Besides landmarks such as positions of bands and centromeres, the short and long arms (denoted p and q arms) of eukaryotic chromosomes have been subdivided into distinct segments that are numbered consecutively, so that numbers indicate distances from centromeres and telomeres.
Hence the positions of binding of labeled probes can be mapped precisely. The resolving power of this technique is not sufficient to distinguish between two genes that are about 5 centiMorgans (5 cM) apart.
The cloned DNA probes used in situ hybridisation are labelled radioactively or by fluorochromes. When the probe is radiolabeled, the positions in denatured chromosomes at which the probe is hybridised are determined by autoradiography.
That is, the chromosome preparations on a glass slide are covered with a film or a thin layer of liquid emulsion containing silver bromide (AgBr) crystals, kept in dark for 2 to 3 weeks to allow radioactive emissions from the probe to reduce the AgBr. Developing the film washes out unreduced grains and shows dark spots where the probe has hybridised.
When the probe is labeled using a fluorescent dye, the procedure is called fluorescent in situ hybridisation (FISH). The cloned DNA is labelled with a fluorescent dye and the chromosome preparation is immersed in the dye containing the probe. When the probe has hybridised to the chromosomes, the slide is scanned in a fluorescence or a laser confocal microscope, and images of fluorescent spots are recorded.
Another procedure that is a variation of FISH is called chromosome painting. This technique uses a standard control set of probes that are homologous to known locations in order to prepare a cytogenetic map. Sets of cloned DNA sequences that are known to be from specific chromosomes or from specific chromosome regions are used as probes. Each set of cloned DNA is labeled with a different fluorescent dye.
When the probes have hybridised, each with its homologous region in denatured chromosomes, the fluorescent dyes “paint” specific regions, they are identified microscopically on the basis of colors. This procedure can include a probe consisting of a cloned sequence of unknown location, labelled with another dye. Its position would be indicated by its dye.
Radiation Hybrid Mapping:
The technique of radiation hybrid mapping is used to develop a high resolution map of molecular markers along a chromosome. It is based on the principle that when cells from two cell lines in culture, one from human the other from rodent, are fused with one another, the resulting hybrid cells retain only a few human chromosomes, the remainder are eliminated during cell divisions.
The human chromosomes that are retained are inherited by descendents of the hybrid cell, and represent a clone of cells. The selection of human chromosomes that would be retained in the hybrid cell seems to occur in a random manner.
For mapping studies in radiation hybrids, instead of whole chromosomes, each hybrid cell line contains a random set of human chromosome fragments (Fig. 25.1). Fragmentation is accomplished by irradiating human cells in culture with a lethal dose of X-rays (3000 rads). The irradiated cells are then fused with cultured rodent cells to form somatic hybrid cells whose nuclei are also fused (heterokaryons).
These are cloned in wells of tissue culture plate, a series of clones is obtained, each containing a different random assortment of human chromosome fragments. From these a radiation hybrid mapping panel can be made.
The fragments of human chromosomes integrate into rodent chromosomes because of breaks produced by X-rays, followed by joining. Cytological examination of cells shows that some rodent chromosomes have integrated fragments, and some contain whole chromosomes of humans.
The integrated fragments are found to display banding patterns that correspond with chromosomes of irradiated human cells. Cell lines with single inserts and identifiable banding patterns are chosen. They are made to overlap one another to make a panel of radiation hybrids representing the whole genome.
DNA is isolated from each cell line in the radiation hybrid mapping panel, placed separately on membranes and denatured. A labeled single copy human DNA probe is hybridised to the membranes. Position of the label identifies the cell line carrying a human chromosome fragment homologous to the probe. The next step is to analyse data on probe hybridisation to detect co-retention of DNA markers.
If two DNA markers map near each other on the same chromosome, it means they have been co-retained. In the mapping panel, higher the frequency of co-retention of two human DNA markers, the closer the two markers are, in map distance, on the same human chromosome. The co-retention of different human markers in radiation hybrid mapping panels allows high resolution mapping of the DNA markers on chromosomal loci.
The radiation hybrid mapping method has an advantage. A standard panel of only about 100 to 200 hybrids is sufficient to generate a high resolution map of the human genome.
Physical Mapping of the Genome:
The genetic maps developed from meiotic recombination studies, described above, do not have sufficient resolution to allow sequencing of the genome. It is therefore, necessary to generate a detailed physical map. The goal is to have a map of markers based on direct analysis of genomic DNA, instead of analysis of recombinants from pedigree analysis.
Physical maps are maps of cloned genomic DNA, that are supplements of a genetic map. There are 24 physical maps, corresponding to the 22 autosomes, an X and a Y chromosome in humans. In situ hybridisation, FISH and radiation hybrid mapping methods provide higher resolution physical maps of human genome. We now describe restriction mapping and clone contig methods for generating physical maps.
Restriction Mapping:
When the entire genome has to be mapped by using restriction enzymes, the commonly used enzymes such as EcoR I and Hind III present serious limitations. These enzymes have a very large number of restriction sites throughout the genome and yield too many fragments that would produce a smear on the gel and cannot be resolved separately.
Therefore, two types of enzymes are used, enzymes that recognise 7 to 8 nucleotide sequences, and enzymes that have recognition sequences that are uncommon in the DNA being mapped.
For example, enzyme Not I can be used which has recognition sequence:
5′-GCGGCCGC-3′
3′-CGCCGGCG-5′
Not I cuts human DNA once every 10 Mb on an average. The second type of enzymes recognise sequences that are uncommon, being present in low frequencies in human DNA. For example, the sequence 5′-GC-3′ and its complementary 3′-CG-5′ are rare in the human genome.
Therefore, enzymes that have 5′-GC-3′ and 3′-CG-5′ in their recognition sequences can cut human DNA less frequently because it is rare. Restriction maps made with these types of restriction enzymes are able to resolve genes that are several hundred kilo-base pairs apart.
However, even with the availability of enzymes like Not I, generating restriction maps of all the chromosomes is an uphill task. In year 2000, restriction map of one of the smallest human chromosomes, that is chromosome number 21, was the only one to have been completed by this method.
Clone Contig Map:
A clone contig map (contig is a short form of contiguous) comprises a set of ordered (occurring consecutively) partially overlapping clones comprising all the DNA of an individual chromosome without any gaps. It is necessary to construct 24 contig maps, one each for the 22 autosomes, an X and a Y chromosome.
Because of the large size of the human genome, vectors that can accommodate large-sized DNA inserts only can be used. Otherwise the number of clones would become enormous, and difficult to handle. To start with, YAC vectors which can carry several hundred kilo-base DNA inserts were used.
The construction of a clone contig map begins with the preparation of a library of partially overlapping DNA fragments. Either whole genomic DNA is used, or individual chromosomes are first separated by flow cytometry before DNA is isolated. Usually the DNA is sheared mechanically by passing through a syringe needle to procure a complete set of randomly overlapping fragments of genomic DNA.
This method of shearing produces blunt ends, therefore, to insert these fragments into YAC vector, the restriction enzyme used for cutting must be one that produces blunt ends, such as SmaI. By this method a YAC library of either whole genomic DNA is produced, or a library for a particular chromosome.
The YAC clones can yield contig maps by using chromosome mapping techniques such as FISH. For large genomic regions, DNA fingerprinting is used. The clones are assembled in order on the basis of overlaps between them. However, the most commonly used method involves sequence-tagged site (STS) method. An STS which serves as a DNA sequence marker is a short unique sequence in the genome that can be amplified by using defined PCR primers.
The technique is based on the principle that clones which share STSs must overlap each other. PCR will screen individually all the clones from a YAC library. All clones containing a particular STS amplified by the primer pair used will yield amplification product. Samples of the DNA in the PCR reaction mixtures for each clone after STS amplification are examined for labelled DNA products using fluorescent or radiolabelling or by techniques that detect complementarity.
If the library is complete with no gaps, it would produce a complete contig map for the chromosome or genomic region being assembled, by locating individual STSs on the genome map on the basis of their locations on cloned DNA. YAC mapping has been done successfully for chromosome number 21.
Besides YAC contig maps, BAC cloning vectors have been used and they have provided more accurate clone contig maps. HGP team has also used radiation hybrid mapping of STS markers and prepared a map with 15,806 STS markers. These maps were further enhanced by using other methods, one of which is expressed sequence tag (EST) method which added an additional 20,104 STS markers.
This involves PCR using oligonucleotide primers designed on the basis of the sequence of a complementary DNA (cDNA), to obtain an EST marker as a PCR product. A cDNA is complementary to the mRNA transcript, therefore, an EST marker corresponds to a functional protein coding gene. If the gene is a unique one, then the EST of that gene is also unique. By determining EST markers, the map was enriched with a large number of protein coding genes.
Individual Chromosome Libraries:
The construction of a physical map of the whole genome presents many challenges even with the use of STS markers. There may be identical sequences, mega base in size, whose duplicate copies may exist in two different chromosomes.
Or there may be gaps in the physical map due to certain regions in the genome that do not clone efficiently. To overcome these difficulties, individual chromosomes can be isolated for preparation of chromosome specific libraries and to develop maps of DNA in each chromosome.
To isolate individual chromosomes if they are small as in yeast, pulse field gel electrophoresis (PFGE) can be used. For large chromosomes in the human genome, fragments have to be prepared by using restriction enzyme Not I which recognises an eight base pair sequence and cuts only once in every 64,000 base pairs. The large sized fragments are separated by PFGE.
Flow Sorting of Chromosomes:
Specific human chromosomes can be separated (flow- sorted) by fluorescence-activated chromosome sorting (FACS). Whole metaphase chromosomes are isolated into a suspension by disrupting dividing cells. The metaphase chromosomes are stained with two fluorescent dyes, one which binds to AT-rich regions, the other to GC-rich regions. The technique is based on the principle that every chromosome has a characteristic ratio of AT-rich to GC-rich regions.
The suspension of stained chromosomes is diluted to an appropriate concentration, so that when converted into a spray of droplets, each droplet contains one chromosome. The droplets flow in a tube under a laser beam that excites fluorescence. The computerized detection system sorts out each chromosome according to the ratio of the AT-rich to GC-rich regions, and collects each chromosome in an individual tube.
DNA from individual chromosomes is isolated and used for preparing chromosome-specific libraries containing inserts from these DNAs. Chromosomes are identified by hybridisation with probes known to be complementary to each of the chromosomes. Chromosome libraries are used to map STS or other markers to develop physical maps.
Sequencing the Genome:
The construction of a high resolution map leads to the next step of sequencing the genome. The dideoxy method of sequencing is used. However, for the large-sized inserts in BAC vectors used for the human genome, a more suitable method of whole genome shotgun sequencing is used.
In the shotgun approach, each insert is cut out and sheared mechanically into a partially overlapping set of fragments (Fig. 25.2) and cloned into a plasmid vector. These sub clones are then sequenced, and the overlapping sequences are assembled into a contiguous sequence using computational methods.
Each BAC clone is mapped onto the chromosome. To assemble the complete sequence of one chromosome, sequences for the individual clone inserts are integrated into one contiguous sequence. The sequences of all the chromosomes then constitute the whole genomic sequence.
Optical Mapping of Whole Genome:
Optical mapping is a procedure for constructing whole genome restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. A key element of optical mapping system is that the DNA molecules are elongated and immobilised on positively charged glass surfaces and subsequently cut with a restriction endonuclease.
The procedure for optical mapping begins with extraction of DNA from any source. The extracted DNA is applied to a microfluidic device called optical chip that consists of a glass surface bearing a mask of several channels. As the DNA sample flows through the multiple channels of the optical chip, individual DNA molecules elongate and become fixed (immobilised) to the surface.
Immobilisation occurs via electrostatic interactions between the negatively charged DNA and positively charged glass surface. The individual DNA molecules range in size from 0.5 to 2.5 million base pairs. Fragments that are 2 kilo-base or smaller may be difficult to retain on the glass surface and could be lost, resulting in errors during image acquisition and subsequent processing.
The array of all the DNA molecules can be examined for the presence of specific markers. One optical chip is capable of capturing multiple copies of a genome from any source, microbe to man.
Subsequent to immobilisation of the DNA molecules, cutting with a restriction endonuclease is carried out. Typically, restriction enzymes that recognise a 6 base pair sequence are used to cleave the DNA. Since the elongated DNA molecules are under slight tension, their ends retract, resulting in gaps. The gaps are visualised after staining with a fluorescent dye.
The generation of optical images requires a computerised optical mapping station mounted on a vibration-free table. There is a computerised argon ion laser scanning fluorescence microscope in the work station that scans the optical chip and processes the information to generate visual images of each field. The workstations are configured for groups of six stations.
The optical mapping system takes over the images produced by each station. It has a multiprocessing data analysis system called the optical mapping cluster. This system integrates the overlapping visual fields and selects molecules for analysis. Single molecule maps are then constructed from the order and size of the fragments.
The mass of the fragments is determined by the automated image analysis software which locates each fragment and measures the amount of fluorescent dye bound to it. The gaps at the ends of the DNA fragments, their presence and spacing, also serve as markers and are typical for each sample.
In optical mapping, any given region of the genome is represented by a number of different DNA molecules which are seen in the form of individual concentric rings in the sample. Overlapping sets of single molecules can be assembled out of the concentric rings by using proprietary software’s that can search similar fragment patterns in different single molecules and develop them into a consensus map representing the whole genome.
Ethical, Legal and Social Issues (ELSIs):
Scientists and general public have expressed their concerns concerning potential use of new genomic information. The following ethical, legal and social issues (ELSIs) are under examination.
These include privacy and confidentiality of genetic information; fairness in use of genetic information by employers, insurance companies and others; psychological impact of genetic deviations on individuals; reproductive and clinical issues; environmental issues concerning genetically modified microbes.
Synthetic Genomics:
An emerging new technology involving genome transplantation allows one type of bacterium to become transformed into another type. The transformed bacterium then expresses information contained in the transplanted chromosome. Thus, scientists Carole Lartigue and colleagues (2007) have been able to change Mycoplasma capricolum into Mycoplasma mycoides Large colony (LC). Mycoplasmas are small cells lacking a cell wall.
The procedure for genome transplantation is carried out in several steps. First, a marker gene is incorporated into M mycoides LC chromosome. The marker gene is antibiotic selectable, therefore, it allows selection of living M. mycoides LC cells containing the transplanted chromosome. In the next step, DNA (chromosome) is extracted from the selected M. mycoides LC cells and purified to eliminate proteins.
This chromosome of M. mycoides is then transplanted into cells of M. capricolum. Repeated proliferation of M capricolum cells resulted in elimination of the recipient M. capricolum chromosome, and it was replaced by the M. mycoides LC chromosome. The transplanted M. mycoides LC chromosome was expressed, and M. capricolum cells displayed all the phenotypic characteristics of M. mycoides LC cells.
Results of the above experiments were confirmed using gel electrophoresis and protein sequencing. The expressed proteins were found to be the ones coded for by the M. mycoides LC chromosome.
Subsequent to the success of the transplant experiments, a future goal of synthetic genomics is to attempt transplantation of chemically synthesised chromosomes into viable living cells. The important applications of synthetic genomic research include development of new energy sources, pharmaceuticals and chemicals.