The sequence of the human genome. Science , — All rights reserved. In the whole-genome assembly method also called the whole-genome random shotgun method , Celera generated a massive shotgun library derived from its own DNA sequence data combined with the "shredded" Human Genome Project DNA sequence data, which together corresponded to a total of Celera used computational methods and sophisticated algorithms to identify overlapping DNA sequences and to reconstruct the human genome by generating a set of scaffolds Figure 5.
In contrast, with the regional chromosome assembly approach also called the compartmentalized shotgun assembly method , Celera organized its own data and the Human Genome Project sequence data into the largest possible chromosomal segments, followed by shotgun assembly of the sequence data within each segment Venter et al.
The first step of the regional assembly approach involved separating Celera reads that matched Human Genome Project reads from those that were distinct from the public sequence data. Of the These reads were assembled into Celera-specific or Human Genome Project-specific scaffolds, which were then combined and analyzed using whole-gene assembly algorithms.
The resulting bactig data were again "shredded" to permit unbiased assembly of the combined sequence data. Celera's whole-genome and regional chromosome assembly methods were independent of each other, permitting direct comparison of the data. Celera found that the regional chromosome assembly method was slightly more consistent than the whole-genome assembly method. In February , drafts of the human genome sequence were published simultaneously by both groups in two separate articles IHGSC, ; Venter et al.
Due to technical advances in DNA sequencing methods and a productive level of synergy between the two groups, they tied at the finish line , and both projects were completed ahead of schedule. As previously mentioned, the IHGSC and Celera used different approaches to determine the sequence of the human genome.
The mixture was first heated to denature the template DNA strand; this was followed by a cooling step to allow the DNA primer to anneal. Following primer annealing, the polymerase synthesized a complementary DNA strand. The template would grow in length until a dideoxynucleotide base ddNTP was incorporated; the conditions were such that this occurred at random along the length of the newly synthesized DNA strands.
In order to determine the sequence of the newly synthesized, color-coded DNA strands, researchers needed a way to separate them based on their size, which differed by only one DNA nucleotide. To accomplish this, they electrophoresed the DNA through a gel matrix that permitted single-base differences in size to be easily distinguished.
Small fragments run more quickly through the gel, and larger fragments run more slowly Figure 6c. By putting the entire mixture into a single well of the gel, a laser can be used to scan the DNA bands as they move through the gel and determine their color; this data can be used to generate a sequence trace also called an electropherogram , showing the color and signal intensity of each DNA band that passes through the gel Figure 6d.
Unfortunately, the initial hope of accelerating the discovery of new treatments for disease was not necessarily accomplished by the Human Genome Project. With the sequence of the human genome in hand, we have learned that it requires more than just knowledge of the order of the base pairs in our genome to cure human disease.
Current efforts are therefore focused on understanding the protein products that are encoded by our genes. When a gene is mutated, the corresponding protein is most often defective. The emerging field of proteomics aims to understand how protein function and expression are altered in human disease states.
Furthermore, investigators are also turning their attention to the expansive regions of our genome devoid of traditional protein-encoding genes. We have already started to reap the benefits of our knowledge of the human genome, and future data-mining efforts will most certainly uncover many more exciting and unexpected links to human disease.
International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature , — link to article. Finishing the euchromatic sequence of the human genome.
Venter, J. Science , — link to article. Pufferfish and Ancestral Genomes. Simple Viral and Bacterial Genomes. Complex Genomes: Shotgun Sequencing. DNA Sequencing Technologies. Genomic Data Resources: Challenges and Promises. Transcriptome: Connecting the Genome to Gene Function. Behavioral Genomics.
Comparative Methylation Hybridization. Pharmacogenomics and Personalized Medicine. Sustainable Bioenergy: Genomics and Biofuels Development. Citation: Chial, H. Nature Education 1 1 Thanks to the Human Genome Project, researchers have sequenced all 3. How did researchers complete this chromosome map years ahead of schedule? Aa Aa Aa. Phases of the Human Genome Project. The total is the sum of finished sequence red and unfinished draft plus predraft sequence yellow.
Nature , Figure Detail. The BAC library is represented by short, disordered, squiggly black line segments. Next, the clones are organized and mapped into overlapping large clone contigs. One of the BAC clones is randomly chosen for sequencing. It is fragmented into small pieces, which are subcloned into vectors to generate shotgun clones.
These clones are then sequenced. Overlapping portions of the shotgun sequences are assembled to determine the genomic sequence. Figure 3: Levels of clone and sequence coverage. Minimally overlapping clones are picked from a fingerprint clone contig for sequencing.
The clones are sequenced to at least draft coverage to form a sequenced-clone contig. The sequences are then merged and ordered to create a sequence-contig scaffold. Celera: Shooting at Random and Organizing Later. Figure 4: Architecture of Celera's two-pronged assembly strategy.
Figure 5: Anatomy of whole-genome assembly. In whole-genome assembly, the BAC fragments red line segments and the reads from five individuals black line segments are combined to produce a contig and a consensus sequence green line. The contigs are connected into scaffolds, shown in red, by pairing end sequences, which are also called mates.
If there is a gap between consecutive contigs, it has a known size. Next, the scaffolds are mapped to the genome gray line using sequence tagged site STS information, represented by blue stars. Figure 6: How to sequence DNA. This step produces a mixture of newly synthesized DNA strands that differ in length by a single nucleotide. C The DNA mixture is separated by electrophoresis. D The electropherogram results show peaks representing the color and signal intensity of each DNA band. From these data, the sequence of the newly synthesized DNA strand is determined, as shown above the peaks.
Dennis, C. Used with permission. Panel B shows nine newly synthesized DNA strands. Each of the strands differs in length by a single nucleotide and is labeled at the 3' end with a fluorescently-labeled ddNTP base. Panel C shows the electrophoresis results. The DNA strands have been separated by size and appear as columns of colored bands.
Panel D shows the electropherogram results, which are a series of colored peaks, with red representing T, black representing G, blue representing C, and green representing A. Shown above the peaks is the DNA sequence. From Rough Draft to Final Form. During this phase, the researchers filled in gaps and resolved DNA sequences in ambiguous areas that were not solved during the shotgun phase.
The final form of the human genome contained 2. Furthermore, the IHGSC reduced the number of gaps by fold; only gaps out of , gaps remained. An entire set of DNA molecules in the nucleus of eukaryotic organisms is called the genome.
DNA has two complementary strands linked by hydrogen bonds between the paired bases. Messenger RNA mRNA is analyzed most frequently because it represents the protein-coding genes that are being expressed in the cell. To study or manipulate nucleic acids, the DNA must first be extracted from cells. Various techniques are used to extract different types of DNA Figure Most nucleic acid extraction techniques involve steps to break open the cell, and then the use of enzymatic reactions to destroy all undesired macromolecules.
Cells are broken open using a detergent solution containing buffering compounds. To prevent degradation and contamination, macromolecules such as proteins and RNA are inactivated using enzymes. The DNA is then brought out of solution using alcohol. The resulting DNA, because it is made up of long polymers, forms a gelatinous mass. RNA is studied to understand gene expression patterns in cells.
Some are even secreted by our own skin and are very difficult to inactivate. Because nucleic acids are negatively charged ions at neutral or alkaline pH in an aqueous environment, they can be moved by an electric field.
Gel electrophoresis is a technique used to separate charged molecules on the basis of size and charge. The nucleic acids can be separated as whole chromosomes or as fragments. The nucleic acids are loaded into a slot at one end of a gel matrix, an electric current is applied, and negatively charged molecules are pulled toward the opposite end of the gel the end with the positive electrode.
Smaller molecules move through the pores in the gel faster than larger molecules; this difference in the rate of migration separates the fragments on the basis of size.
The nucleic acids in a gel matrix are invisible until they are stained with a compound that allows them to be seen, such as a dye. Distinct fragments of nucleic acids appear as bands at specific distances from the top of the gel the negative electrode end that are based on their size Figure A mixture of many fragments of varying sizes appear as a long smear, whereas uncut genomic DNA is usually too large to run through the gel and forms a single large band at the top of the gel.
DNA analysis often requires focusing on one or more specific regions of the genome. It also frequently involves situations in which only one or a few copies of a DNA molecule are available for further analysis. These amounts are insufficient for most procedures, such as gel electrophoresis. Polymerase chain reaction PCR is a technique used to rapidly increase the number of copies of specific regions of DNA for further analyses Figure PCR is used for many purposes in laboratories.
These include: 1 the identification of the owner of a DNA sample left at a crime scene; 2 paternity analysis; 3 the comparison of small amounts of ancient DNA with modern organisms; and 4 determining the sequence of nucleotides in a specific region. In general, cloning means the creation of a perfect replica. Typically, the word is used to describe the creation of a genetically identical copy. Cloning allows for the creation of multiple copies of genes, expression of genes, and study of specific genes.
To get the DNA fragment into a bacterial cell in a form that will be copied or expressed, the fragment is first inserted into a plasmid.
A plasmid also called a vector in this context is a small circular DNA molecule that replicates independently of the chromosomal DNA in bacteria. Modified plasmids are usually reintroduced into a bacterial host for replication.
As the bacteria divide, they copy their own DNA including the plasmids. Plasmids occur naturally in bacterial populations such as Escherichia coli and have genes that can contribute favorable traits to the organism, such as antibiotic resistance the ability to be unaffected by antibiotics. Plasmids have been highly engineered as vectors for molecular cloning and for the subsequent large-scale production of important molecules, such as insulin. A valuable characteristic of plasmid vectors is the ease with which a foreign DNA fragment can be introduced.
These plasmid vectors contain many short DNA sequences that can be cut with different commonly available restriction enzymes. Restriction enzymes also called restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA.
Many restriction enzymes make staggered cuts in the two strands of DNA, such that the cut ends have a 2- to 4-nucleotide single-stranded overhang. The sequence that is recognized by the restriction enzyme is a four- to eight-nucleotide sequence that is a palindrome. Like with a word palindrome, this means the sequence reads the same forward and backward. In most cases, the sequence reads the same forward on one strand and backward on the complementary strand. When a staggered cut is made in a sequence like this, the overhangs are complementary Figure In this way, any DNA fragment can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction enzyme Figure Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they contain new combinations of genetic material.
Proteins that are produced from recombinant DNA molecules are called recombinant proteins. Not all recombinant plasmids are capable of expressing genes. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins. Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism.
Most multicellular organisms undergo reproduction by sexual means, which involves the contribution of DNA from two individuals parents , making it impossible to generate an identical copy or a clone of either parent. Recent advances in biotechnology have made it possible to reproductively clone mammals in the laboratory.
Natural sexual reproduction involves the union, during fertilization, of a sperm and an egg. Each of these gametes is haploid, meaning they contain one set of chromosomes in their nuclei.
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