New Technique reveals unseen information in DNA code

Imagine reading an entire book, but then realizing that your glasses did not allow you to distinguish "g" from "q." What details did you miss?  


Geneticists faced a similar problem with the recent discovery of a "sixth nucleotide" in the DNA alphabet. Two modifications of cytosine, one of the four bases that make up DNA, look almost the same but mean different things. But scientists lacked a way of reading DNA, letter by letter, and detecting precisely where these modifications are found in particular tissues or cell types. The team used the technique to map 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in DNA from human and mouse embryonic stem cells, revealing new information about their patterns of distribution. These studies have revealed that these DNA modifications play major roles in fundamental life processes such as cell differentiation, cancer and brain function. They regulate gene expression and have a broad impact on stem cell development, various human diseases such as cancer, and potentially on neurodegenerative disease, they may even shape the development of the human brain. Geneticists describes a method called TAB-Seq that directly measures 5-hmC, and presents the first map of the entire genome of 5-hmC at single-base resolution. It's a major breakthrough in that TAB-Seq allows precise mapping of all 5-hydroxymethylcytosine sites in a mammalian genome using well-established, next-generation DNA sequencing methods.  The study showed very clearly that deriving useful knowledge about this poorly understood epigenetic regulator requires determination of the exact locations of 5hmC with base-level accuracy. 

Dark Areas of DNA

A massive effort to sequence and compare 29 mammalian genomes has shed new light on the “dark matter” of the genome, the over 98% of DNA that doesn’t code for proteins. The DNA that lies outside of gene sequences was once called “junk DNA.” But researchers now know that these non-coding regions have important biological functions. Many disease-causing mutations have been found in these areas, and scientists have pieced together some clues to their functions. For example, some regions regulate the expression of genes, controlling when genes are turned on and off. Nevertheless, this vast genetic dark matter remains largely uncharted.To gain new insights, an international team of researchers set out to compare the sequences of several mammalian species. Regions that remain the same or have only gradually evolved, they reasoned, must have some function.

In the early online edition of Nature on October 12, 2011, the researchers reported the sequencing of 20 new mammalian genomes, including rabbit, dolphin and elephant. They compared these new sequences with 9 others that were previously described, including humans. The scientists found that at least 5% of the genome appears to be constrained by evolution. They were able to identify 3.6 million specific elements under constraint, which together make up over 4% of the human genome. These elements include hundreds of new families of RNA, thousands of previously undetected segments of protein-coding DNA, and 2.7 million elements thought to play a role in controlling gene expression.

Significantly, the researchers found that many of the elements they identified overlap with variants that were linked to diseases and conditions in previous genomics studies.

What are the advantages of Step one Plus RT-PCR?

The step one plus RT-PCR ( 96 well platform, 4 color system, veriflex sample blocks) use fluorescent-based PCR reagents to provide 1) Quantitative detection of target nucleic acid sequences using real time analysis, 2) Qualitative detection of targets using post-PCR (end point) analsyis, 3) Qualitative analysis of the PCR product (achieved by melt curve analysis that occur post-PCR) and 4) Time saving ( one full run of PCR plate completed within 40minutes ). The step one plus RT-RCR instrument contains 6 independently thermally regulated "veriflex" blocks to help to optimize the thermal cycling conditions. We can set a different temperature for one or more of the veriflex blocks, (or) we can set the same temperature for each of the veriflex blocks. The following experiments can be perform using the step one plus RT-PCR:

• Quantitation - Standard curve
• Quantitation - Relative standard curve
• Quantitation - Comparative CT 
• Melt curve
• Genotyping
• Presence / Absence experiments
• Minimizing DNA contaminants

Following 4 filters can be used by the step one plus RT-PCR : 1) FAM dye - SYBR Green dye, 2) JOE dye - VIC dye, 3) TAMRA dye- NED dye, and 4) ROX dye.

What are the advantages of the operon organization within the bacterial chromosome?

A major advantage is that it leads to the synthesis of groups of functionally related enzymes, usually from a single mRNA transcript. Since a functional pathway must be activated in terms of all of its components, the operon affords an all-or-none response that serves efficiency. The control mechanisms existing for turning on or turning off the operon make the operon sensitive to those changes in the environment to which the bacterial cell must adjust.

The ability of the cell to fashion repressors that are functional as well as repressors that are nonfunctional permits a broad variety of environmental triggers to exert their effects. Inducible systems may be activated by agents that tie up the repressor, and constitutive systems may be shut off by corepressors that activate initially ‘‘blank’’ repressors. Further fine tuning is achieved by the participation of CAP protein and cyclic AMP in the activation of the promoter site.

Suggest the mechanism by which operons control transcription.

In order for DNA to initiate the formation of RNA for subsequent translation, it must first unite with an RNA polymerase. The site for RNA polymerase attachment on the DNA is called the promoter. The operator lies within the promoter region (or overlaps it) so that attachment of the RNA polymerase is blocked when the repressor is present. In the presence of an inducer (lactate or a closely related isomer, in the case of the lac operon), the repressor is inactivated (and thus detaches from or fails to attach to the operator), and the operon turns on in the typical inducible fashion. RNA polymerase may then attach to the promoter site, and transcription can be carried out. Transcription occurs along the DNA template in a 3' to 5' direction. In the case of the lac operon, for example, a singleRNAtranscript is synthesized for the three structural genes of the lac operon. Before translation, processing of this single RNA molecule will occur to permit the generation of three separate enzymes.

How chromosome movements are organized?

The movement phases of chromosomes are designated pro-metaphase, metaphase, and anaphase. During pro-metaphase, the nuclear lamina disintegrates and the nuclear envelope breaks into small vesicles permitting the fibers of the spindle to “invade” the nuclear region. The spindle microtubules then associate with kinetochores. These are called kinetochore microtubules. The microtubules from one pole associate with the kinetochore of one of the members of a pair of chromatids. Microtubules from the other pole associate with the kinetochore of the other member. Repulsive forces from the poles push chromosomes toward the center, or equatorial plate, in a rather aimless back and forth motion. The two chromatids are held together, presumably by proteins called cohesins. During metaphase, the kinetochores arrive at the equatorial plate. Chromosomes are fully condensed and have distinguishable shapes. Cohesins break down. DNA topoisomerase II unravels the interconnected DNA molecules at the centromere, and all the chromatids separate simultaneously. Anaphase begins when the centromeres separate. The process takes 10 to 60 minutes for the chromosomes to move to opposite poles. Molecular motors at the kinetochores move the chromosomes toward the poles, accounting for about 75% of the motion. About 25% of the motion comes from shortening of the microtubules at the poles. Additional distance is gained by the separating of the mitotic centers. This increase in distance between the poles is done by the polar microtubules, which have motor proteins associated in the overlapping regions. By this process the distance between the poles doubles.

Exon shuffling

Another mechanism that explains how the genome can encode for such a wide array of proteins is alterative splicing. Normally RNA splicing removes the introns and then reattaches the exons in sequence. However, in alternative splicing some of the exons may not be reattached. Since the exons contain coding information for the polypeptide, the removal of some of the exons will produce a slightly different form of the protein. It is important to note that the order of the exons is usually not changed, some of them are just not included in the final mRNA. Alternative splicing is also sometimes called exon shuffling. From an evolutionary perspective, alternative splicing allows a species to produce proteins of related function from a single transcription event. Often these proteins are tissue specific, meaning that the pre-mRNA is edited one way in one tissue, but is alternatively spliced in another. There are many examples of alternative splicing in mammals, and it is believed to play a major role in explaining the size of the proteome.

Rates of Mutation

Geneticists use the term mutation rate to indicate the probability that a given nucleotide will be changed by a spontaneous mutation. Mutation rates are expressed as the chance of a mutation per round of replication, per gamete (in eukaryotes), or per cell division (in prokaryotes). Mutation rates also vary considerably between organisms. Bacteria and viruses tend to have higher mutation rates due to the fact that they do not generally possess efficient mechanisms of DNA repair. The mutation rate for humans is 1× 10−5 to 1 × 10 −6 . In other words, a single nucleotide has a chance of mutation once per million to 10 million gametes. Mutation rates are not precise measurements. Rather, they are considered to represent the probability of mutation. Geneticists recognize that mutation rates are not consistent throughout the genome. Areas of the genome that contain repetitive sequences are more prone to mutation than other areas. These are called mutational hot spots, and they have been discovered in the genomes of most organisms. Geneticists who are concerned with mutations at the population level use the term mutation frequency to indicate the rate of a specific mutation within a population. This term does not indicate the chances that the mutation will arise spontaneously within the population, but rather the estimated percent of the population that carries the mutation.

How Genomics formed

Genomics is the study of genes, their structure and function. In 1990, the U.S. Department of
Energy and the National Institutes of Health began the Human Genome Project. The goals of this
project were to determine the sequences of the 3 billion nitrogen base pairs in DNA, to identify
the 30,000 genes in human DNA and to store this information in a database that would be available to researchers. In September 1999, the Celera corporation entered the race to map the
human genome. By April 2000, Celera scientists completed the sequencing of the nitrogen bases and began the mapping phase of genes, which was completed by the end of 2000. The Human Genome Project completed its work in April 2003, about 50 years after Watson and Crick’s discovery of the structure of DNA. The Human Genome Project is important because it will lead to increased understanding of the role of genetics in the maintenance of health and the combat of disease. We hope to enhance our knowledge of gene function and to discover new methods of diagnosing disease. As a result of the Human Genome Project, new drugs and therapeutic measures for the treatment of disease will be developed. The Human Genome Project has produced a new area of genetics called genomics.

History of Nucleus

A cell may be uni or multinucleated. If a multinucleated condition arises due to fusion of cells, it is called syncytium e.g., plasmodium, body of slime moulds, young xylem vessels and if due to repeated nuclear divisions without cytokinesis, it is called coenocytic e.g. Vaucheria, Rhizopus. Nucleus was reported by Robert Brown (1831) in orchid cells. Strasburger (1882) proved that nucleus arises from pre existing nucleus by division. Hertwig and van Beneden showed the role of nucleus in fertilization. Hammerling (1953) by his grafting experiments on Acetabularia (largest unicellular green, marine alga) proved the role of nucleus in heredity, growth and morphology. 1/10th of volume of cell is occupied by nucleus. In a cell, there is a definite nucleo cytoplasmic ratio. Nucleocytoplasmic index is Volume of nucleus/ (Volume of cell – Volume of nucleus). About 10% of nuclear membrane bears simple of compound pores. Nucleus has 80% proteins (65% non histone, Mol. Wt. high, rich in tyrosine and tryptophan, acidic and forms enzymes and helps in RNA transaction. 15% proteins are basic, histone proteins, Mol. Wt. low, rich in lysine and arginine. DNA : histone ratio 1 : 1. Nucleosomes are structural units of chromatin. Term was given by Outdet. A nucleosome is an octamer of histone proteins and has a core of 8 molecules of histone proteins (two each of H2A, H2B, H3, H4) bounded by 13/4 turns of DNA having about 166 base pairs. H1 histone does not form nucleosome. Size of a chromosome varies from 0.5 to 32 μ. Minimum number of chromosomes n = 2 e.g., Haplopappus. Maximum number is 2n = 1262 in Adder’s fern (Ophioglossuin). In animals, minimum number is 2n = 2 in Ascaris sp. And maximum number is 2n = 1600 in Aulacantha and Radiolarians.

Glimpses about codon?

Initiation codon (i) This is the triplet codon at which polypeptide synthesis begins. (ii) It is always AUG and codes for a methionine. (iii) As a result, all polypeptides are synthsized with an amino-terminal methionine. Coding region (i) This is the sequence of mRNA that contains the consecutive triplet codons that direct polypeptide synthesis. (ii) This region spans from the start codon to the stop codon. (iii) The coding region is often referred to as the open reading frame or ORF. Stop codon (i) This is the triplet codon that signals the termination of translation. (ii) There are three possible stop codon sequences: UAA, UAG, UGA. (iii) Stop codons have no corresponding tRNA or amino acid.

Why Restriction Map is needed?

Any double-stranded DNA will be cut by a variety of restriction enzymes that have different recognition sequences. By separating the restriction fragments and measuring their sizes by gel electrophoresis, it is possible to deduce where on the DNA molecule each restriction enzyme cuts. A restriction map of the DNA molecule can be drawn showing the location of these cut sites (restriction sites). It is then easy to compare two DNA molecules (for example, to examine the evolutionary relationship between two species) by looking at their restriction maps without the need to determine the nucleotide sequence of each DNA. Restriction maps are also important experimentally during recombinant DNA work, both to plan where individual DNA molecules should best be cut and to monitor the progress of the experiment.

What are tools in the Home of Translation?

The ribosome is the factory that produces proteins. Thousands of ribosomes are present in even the simplest of cells. They are complex units composed of RNA and protein. It is possible to dissociate a prokaryotic ribosome into two units. One unit is the 50S, or large unit, and the other is the 30S, or small unit. The large unit contains 34 different proteins, labeled L1 through L34, and two RNA molecules, labeled 23S and 5S. The small unit contains 21 different proteins, labeled S1 through S21, and an RNA molecule labeled 16S. The RNA
molecules in the ribosomes act as transfer RNA in translation. A prokaryotic ribosome contains three rRNA molecules (23S, 16S, and 5S), one copy of proteins S1 through S21, two copies of L7 and L12, and one each of the other L1-L34 proteins. L7 and L12 are identical except that L7 has an acetylated amino terminus. S20 and L26 are identical. Mixing the constituents in vitro leads to the two subunits reconstituting themselves.

Why Histones are required to associate DNA?

Eukaryotic DNA has a group of proteins associated with it. These small, basic proteins are called histones. They are basic because approximately 25 percent of the amino acid residues present are either arginine or lysine. These are tightly bound to the DNA and total approximately half of the mass of a chromosome. A complex of the cell’s DNA and associated protein is a chromatin, and there are five important histones present in chromatin: H1 — and four that associate with each other: H2A, H2B, H3, and H4. A chromatin apparently consists of repeat units consisting of two copies each of H2A, H2B, H3, and H4, with a strand of DNA consisting of about 200 base pairs tightly wrapped around this histone octomer. Each of these repeating units is a nucleosome. The wrapping of the DNA to form a nucleosome yields a significant compaction of the DNA. Research indicates that about 145 of the 200 base pairs are actually associated with the histone octomer, and the remaining base pairs are linker DNA that link one histone octomer to the next. Histone H1 usually binds to linker DNA. The eight histones in a histone octomer are arranged into a tetramer with the composition (H3)2(H4)2 and two dimers each with the composition (H2A)(H2B). All the histone proteins have long tails rich in arginine and lysine residues that extend out of the coreModification of these tails is important for gene regulation. The structure of chromatin is a factor in eukaryotic gene regulation. For a gene to be available for transcription, the tightly packed chromatin structure must open.

How much will it cost to synthesize AMP and GMP?

The biosynthesis of both AMP and GMP requires the hydrolysis of several high-energy bonds. To produce IMP from D-ribose 5-phosphate requires the hydrolysis of five high-energy bonds (one PPi and five ATP). To convert IMP to AMP requires the hydrolysis of one more high-energy bond (from GTP). And to convert IMP to GMP requires the hydrolysis of two high-energy bonds — one ATP and one PPi. Anaerobic organisms, such as the bacteria responsible for tetanus or botulism, must oxidize four glucose molecules at two ATP per glucose to meet the energy requirement. An aerobic organism, like you, for example, needs to oxidize only one glucose molecule at 36 or 38 ATP per glucose. The preceding processes require a substantial amount of energy. Sometimes this energy requirement may be lessened by metabolic processes known as the salvage pathways. In the salvage pathways, nitrogen bases are recycled instead of synthesized. The nitrogen bases are then converted to nucleotides.

Note on Representative gene libraries

A gene library is a collection of different DNA sequences from an organism each of which has been cloned into a vector for ease of purification, storage and analysis. There are essentially two types of gene library that can be made depending on the source of the DNA used. If the DNA is genomic DNA, the library is called a genomic library. If the DNA is a copy of an mRNA population, that is cDNA, then the library is called a cDNA library. When producing a gene library, an important consideration is how well it represents the starting material, that is does it contain all the original sequences (a representative library)? If certain sequences have not been cloned, for example repetitive sequences lacking restriction sites , the library is not representative. Likewise, if the library does not contain a sufficient number of clones, then it is probable that some genes will be missing. cDNA libraries that are enriched for certain sequences will obviously lack others, but if correctly prepared and propagated they can be representative of the enriched mRNA starting material.

What is Direct gene transfer?

Genes may be transiently or permanently introduced into cultured eukaryotic transfer cells without the use of a vector in the strict sense. A eukaryotic gene on a bacterial plasmid, for example, may transiently express its product when transfected into a cell line, even if the plasmid does not replicate in that type of cell. Alternatively, DNA introduced by transfection or microinjection may become stably integrated into the cell’s chromosomal DNA. This process normally requires significant sequence similarity between the incoming DNA and the genome in animal cells but, in plant cells, any supercoiled plasmid can randomly integrate into the genome in a process which is not understood in detail. Such stably transfected cells can be selected by the presence of a drug resistance gene in much the same way as bacterial transformants, and can continue to express protein from foreign genes through many cell divisions.

How YAC vectors are differentiated?

The realization that the components of a eukaryotic chromosome that are required for stable replication and segregation, at least in the yeast Saccharomyces cerevisiae, consist of rather small and well-defined sequences has led to the construction of recombinant chromosomes (yeast artificial chromosomes; YACs). These were used initially for investigation of the maintenance of chromosomes, but latterly as vectors capable of carrying very large cloned fragments. The centromere, telomere and replication origin sequences have been isolated and combined on plasmids constructed in E. coli. The method of construction of the YAC clone is similar to that for cosmids, in that two end fragments are ligated with target DNA to yield the complete chromosome, which is then introduced (transfected) into yeast cells. YAC vectors can accommodate genomic DNA fragments of more than 1 Mb, and hence can be used to clone entire human genes, such as the cystic fibrosis gene, which is 250 kb in length. YACs have been invaluable in mapping the large-scale structure of large genomes, for example in the Human Genome Project

What are Lysogens?

The lysogenic phase of infection is also used in cloning technology. Genes or foreign sequences may be incorporated essentially permanently into the genome of E. coli by integration of a vector containing the sequence of interest. One example of the use of this method is a strain used for overexpression of proteins by the T7 method. The strain BL21(DE3) and derivatives include the gene for T7 RNA polymerase under control of the lac promoter as a lysogen, designated DE3. The gene can be induced by IPTG, and the polymerase will then transcribe the target gene in the expression vector

How DNA Vaccines are performed?

DNA vaccines are at present experimental, but hold promise for future therapy since they will evoke both humoral and cell-mediated immunity, without the dangers associated with live virus vaccines. The gene for an antigenic determinant of a pathogenic organism is inserted into a plasmid. This genetically engineered plasmid comprises the DNA vaccine which is then injected into the host. Within the host cells, the foreign gene can be expressed (transcribed and translated) from the plasmid DNA, and if sufficient amounts of the foreign protein are produced, they will elicit an immune response.