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5.9 HUMAN GENOME PROJECT In the preceding sections you have learnt that it is the sequence of bases in DNA that determines the genetic information of a given organism. In other words, genetic make-up of an organism or an individual lies in the DNA sequences. If two individuals differ, then their DNA...

5.9 HUMAN GENOME PROJECT In the preceding sections you have learnt that it is the sequence of bases in DNA that determines the genetic information of a given organism. In other words, genetic make-up of an organism or an individual lies in the DNA sequences. If two individuals differ, then their DNA sequences should also be different, at least at some places. These assumptions led to the quest of finding out the complete DNA sequence of human genome. With the establishment of genetic engineering techniques where it was possible to isolate and clone any piece of DNA and availability of simple and fast techniques for determining DNA sequences, a very ambitious project of sequencing human genome was launched in the year 1990. Human Genome Project (HGP) was called a mega project. You can imagine the magnitude and the requirements for the project if we simply define the aims of the project as follows: Human genome is said to have approximately 3 x 109 bp, and if the cost of sequencing required is US \$ 3 per bp (the estimated cost in the beginning), the total estimated cost of the project would be approximately 9 billion US dollars. Further, if the obtained sequences were to be stored in typed form in books, and if each page of the book contained 1000 letters and each book contained 1000 pages, then 3300 such books would be required to store the information of DNA sequence from a single human cell. The enormous amount of data expected to be generated also necessitated the use of high speed computational devices for data storage and retrieval, and analysis. HGP was closely associated with the rapid development of a new area in biology called Bioinformatics. Goals of HGP Some of the important goals of HGP were as follows: 102 (i) Identify all the approximately 20,000-25,000 genes in human DNA; (ii) Determine the sequences of the 3 billion chemical base pairs that make up human DNA; (iiii) Store this information in databases; (iv) Improve tools for data analysis; (v) Transfer related technologies to other sectors, such as industries; (vi) Address the ethical, legal, and social issues (ELSI) that may arise from the project. The Human Genome Project was a 13-year project coordinated by the U.S. Department of Energy and the National Institute of Health. During the early years of the HGP, the Wellcome Trust (U.K.) became a major partner; additional contributions came from Japan, France, Germany, China and others. The project was completed in 2003. Knowledge about the effects of DNA variations among individuals can lead to revolutionary new ways to diagnose, treat and someday prevent the thousands of 2024-25MOLECULAR BASIS OF INHERITANCE disorders that affect human beings. Besides providing clues to understanding human biology, learning about non-human organisms DNA sequences can lead to an understanding of their natural capabilities that can be applied toward solving challenges in health care, agriculture, energy production, environmental remediation. Many non-human model organisms, such as bacteria, yeast, Caenorhabditis elegans (a free living non-pathogenic nematode), Drosophila (the fruit fly), plants (rice and Arabidopsis), etc., have also been sequenced. Methodologies : The methods involved two major approaches. One approach focused on identifying all the genes that are expressed as RNA (referred to as Expressed Sequence Tags (ESTs). The other took the blind approach of simply sequencing the whole set of genome that contained all the coding and non-coding sequence, and later assigning different regions in the sequence with functions (a term referred to as Sequence Annotation). For sequencing, the total DNA from a cell is isolated and converted into random fragments of relatively smaller sizes (recall DNA is a very long polymer, and there are technical limitations in sequencing very long pieces of DNA) and cloned in suitable host using specialised vectors. The cloning resulted into amplification of each piece of DNA fragment so that it subsequently could be sequenced with ease. The commonly used hosts were bacteria and yeast, and the vectors were called as BAC (bacterial artificial chromosomes), and YAC (yeast artificial chromosomes). The fragments were sequenced using automated DNA sequencers that worked on the principle of a method developed by Frederick Sanger. (Remember, Sanger is also credited for developing method for determination of amino acid sequences in proteins). These sequences were then arranged based on some overlapping regions present in them. This required generation of overlapping fragments for sequencing. Alignment of these sequences was humanly not possible. Therefore, specialised computer based programs were developed (Figure 5.15). These sequences were subsequently annotated and were assigned to each chromosome. The sequence of chromosome 1 was completed only in May 2006 (this was the last of the 24 human chromosomes -- 22 autosomes and X and Y -- to be Figure 5.15A representative diagram of human genome project 103 103 2024-25BIOLOGY sequenced). Another challenging task was assigning the genetic and physical maps on the genome. This was generated using information on polymorphism of restriction endonuclease recognition sites, and some repetitive DNA sequences known as microsatellites (one of the applications of polymorphism in repetitive DNA sequences shall be explained in next section of DNA fingerprinting). 5.9.1 Salient Features of Human Genome Some of the salient observations drawn from human genome project are as follows: (i) The human genome contains 3164.7 million bp. (ii) (iii) The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene being dystrophin at 2.4 million bases. The total number of genes is estimated at 30,000--much lower than previous estimates of 80,000 to 1,40,000 genes. Almost all (99.9 per cent) nucleotide bases are exactly the same in all people. (iv) The functions are unknown for over 50 per cent of the discovered genes. (v) Less than 2 per cent of the genome codes for proteins. (vi) Repeated sequences make up very large portion of the human genome. (vii) Repetitive sequences are stretches of DNA sequences that are repeated many times, sometimes hundred to thousand times. They are thought to have no direct coding functions, but they shed light on chromosome structure, dynamics and evolution. (viii) Chromosome 1 has most genes (2968), and the Y has the fewest (231). (ix) Scientists have identified about 1.4 million locations where singlebase DNA differences (SNPs -- single nucleotide polymorphism, pronounced as 'snips') occur in humans. This information promises to revolutionise the processes of finding chromosomal locations for disease-associated sequences and tracing human history. 5.9.2 Applications and Future Challenges 104 Deriving meaningful knowledge from the DNA sequences will define research through the coming decades leading to our understanding of biological systems. This enormous task will require the expertise and creativity of tens of thousands of scientists from varied disciplines in both the public and private sectors worldwide. One of the greatest impacts of having the HG sequence may well be enabling a radically new approach to biological research. In the past, researchers studied one or a few genes at a time. With whole-genome sequences and new high-throughput technologies, we can approach questions systematically and on a much 2024-25MOLECULAR BASIS OF INHERITANCE broader scale. They can study all the genes in a genome, for example, all the transcripts in a particular tissue or organ or tumor, or how tens of thousands of genes and proteins work together in interconnected networks to orchestrate the chemistry of life. 5.10 DNA FINGERPRINTING As stated in the preceding section, 99.9 per cent of base sequence among humans is the same. Assuming human genome as 3 × 109 bp, in how many base sequences would there be differences? It is these differences in sequence of DNA which make every individual unique in their phenotypic appearance. If one aims to find out genetic differences between two individuals or among individuals of a population, sequencing the DNA every time would be a daunting and expensive task. Imagine trying to compare two sets of 3 × 106 base pairs. DNA fingerprinting is a very quick way to compare the DNA sequences of any two individuals. DNA fingerprinting involves identifying differences in some specific regions in DNA sequence called as repetitive DNA, because in these sequences, a small stretch of DNA is repeated many times. These repetitive DNA are separated from bulk genomic DNA as different peaks during density gradient centrifugation. The bulk DNA forms a major peak and the other small peaks are referred to as satellite DNA. Depending on base composition (A : T rich or G:C rich), length of segment, and number of repetitive units, the satellite DNA is classified into many categories, such as micro-satellites, mini-satellites etc. These sequences normally do not code for any proteins, but they form a large portion of human genome. These sequence show high degree of polymorphism and form the basis of DNA fingerprinting. Since DNA from every tissue (such as blood, hair-follicle, skin, bone, saliva, sperm etc.), from an individual show the same degree of polymorphism, they become very useful identification tool in forensic applications. Further, as the polymorphisms are inheritable from parents to children, DNA fingerprinting is the basis of paternity testing, in case of disputes. As polymorphism in DNA sequence is the basis of genetic mapping of human genome as well as of DNA fingerprinting, it is essential that we understand what DNA polymorphism means in simple terms. Polymorphism (variation at genetic level) arises due to mutations. (Recall different kind of mutations and their effects that you have already studied in Chapter 4, and in the preceding sections in this chapter.) New mutations may arise in an individual either in somatic cells or in the germ cells (cells that generate gametes in sexually reproducing organisms). If a germ cell mutation does not seriously impair individual's ability to have offspring who can transmit the mutation, it can spread to 105 2024-25BIOLOGY the other members of population (through sexual reproduction). Allelic (again recall the definition of alleles from Chapter 4) sequence variation has traditionally been described as a DNA polymorphism if more than one variant (allele) at a locus occurs in human population with a frequency greater than 0.01. In simple terms, if an inheritable mutation is observed in a population at high frequency, it is referred to as DNA polymorphism. The probability of such variation to be observed in noncoding DNA sequence would be higher as mutations in these sequences may not have any immediate effect/impact in an individual's reproductive ability. These mutations keep on accumulating generation after generation, and form one of the basis of variability/polymorphism. There is a variety of different types of polymorphisms ranging from single nucleotide change to very large scale changes. For evolution and speciation, such polymorphisms play very important role, and you will study these in details at higher classes. The technique of DNA Fingerprinting was initially developed by Alec Jeffreys. He used a satellite DNA as probe that shows very high degree of polymorphism. It was called as Variable Number of Tandem Repeats (VNTR). The technique, as used earlier, involved Southern blot hybridisation using radiolabelled VNTR as a probe. It included (i) isolation of DNA, (ii) digestion of DNA by restriction endonucleases, (iii) separation of DNA fragments by electrophoresis, 106 (iv) transferring (blotting) of separated DNA fragments to synthetic membranes, such as nitrocellulose or nylon, (v) hybridisation using labelled VNTR probe, and (vi) detection of hybridised DNA fragments by autoradiography. A schematic representation of DNA fingerprinting is shown in Figure 5.16. The VNTR belongs to a class of satellite DNA referred to as mini-satellite. A small DNA sequence is arranged tandemly in many copy numbers. The copy number varies from chromosome to chromosome in an individual. The numbers of repeat show very high degree of polymorphism. As a result the size of VNTR varies in size from 0.1 to 20 kb. Consequently, after hybridisation with VNTR probe, the autoradiogram gives many bands of differing sizes. These bands give a characteristic pattern for an individual DNA (Figure 5.16). It differs from individual to individual in a population except in the case of monozygotic (identical) twins. The sensitivity of the technique has been increased by use of polymerase chain reaction (PCRyou will study about it in Chapter 9). Consequently, DNA from a single cell is enough to perform DNA fingerprinting analysis. In addition to application in forensic science, it has much wider application, such as 2024-25MOLECULAR BASIS OF INHERITANCE Figure 5.16 Schematic representation of DNA fingerprinting: Few representative chromosomes have been shown to contain different copy number of VNTR. For the sake of understanding different colour schemes have been used to trace the origin of each band in the gel. The two alleles (paternal and maternal) of a chromosome also contain different copy numbers of VNTR. It is clear that the banding pattern of DNA from crime scene matches with individual B, and not with A. in determining population and genetic diversities. Currently, many different probes are used to generate DNA fingerprints