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Wednesday, 21 May 2014

Biochip

A biochip is a device that has some of the features of a computer chip but, instead of doing calculations, it uses living cells or molecules from living cells to greatly speed up certain laboratory tests. A typical biochip is a glass or plastic chip or tile a few inches on a side. It has hundreds or even tens of thousands of microscopic droplets of material stuck to its surface like gum on a sidewalk. A computer looks at the chip using a camera. Information from a biochip can be used to learn about the differences between genes, cells, or drugs. It can also be used to study many other questions about cells. Biochips are also called microarrays, where micro means ‘‘small’’ and an array is any regular grid, such as a chessboard. The droplets on a biochip are laid down in a checkerboard pattern. A square chip five inches on a side may have 40,000 or more spots on its surface.

The most common kind of biochip is the DNA microarray, also called a gene chip or DNA chip. Deoxyribonucleic acid (DNA) is the long, coded molecule used by all living things to pass on traits to offspring. DNA also tells each cell how to make all the molecules it needs to live, like a cookbook containing many recipes. The DNA of almost every living thing is at least slightly different from that of every other.

In one type of DNA chip, genes (short pieces of DNA that code for single molecules) are placed on the chip. Since even large molecules are too small to see with the naked eye, millions of copies of each gene can be placed on a tiny spot on the chip.



HOW DNA CHIP WORKS?




In a DNA chip, each separate spot (also called a probe) contains one type of defective gene. To find out if a person has any of these defective genes in their own DNA, DNA is taken from the person’s cells. Copies of the person’s DNA are made, and these copies are labeled, meaning that they include a chemical that glows when ultraviolet light (which is invisible to the eye) shines on it. Small drops of liquid containing labeled copies of the person’s DNA are then added to the spots on the biochip.

A normal DNA molecule is shaped like a ladder, but the DNA copies being mixed on the biochip are one-sided copies, like a ladder that has been sawed in half lengthwise, cutting every rung in half. When two pieces of one-sided DNA that have matching rungs (or bases, as they are called) meet, they lock or zip together. When this happens, the two pieces of DNA are said to hybridize. If the patient’s genes match any of the defective genes that have been put on the biochip, they will attach to (hybridize with) those defective genes.

The chip is then washed to remove any of the person’s DNA that has not found a match on the chip. Finally, the chip is placed in ultraviolet light, and a camera records any spots that glow. These are spots where the labeled copies of the person’s DNA have matched up with DNA on the chip.

Examining a patient’s DNA for defects is called genetic screening. By using a biochip, genetic screening can be done very quickly, all the tests can be done at once, rather than doing hundreds or even thousands of separate tests.

Genetic screening is only one way of using biochips. Another important use for biochips is to study how genes are used by living cells. Each gene tells the cell how to make a certain protein molecule. Cells read the recipe given by the gene by first making another molecule, mRNA, which copies the information in the gene. The mRNA can then go to a place in a cell that will build the molecule that the gene codes for. The more mRNA a cell has for a gene at a particular time, the more it is said to be ‘‘expressing’’ that gene, i.e, the more of that particular molecule it is making. Gene expression changes all the time for thousands of genes in every cell.

In the laboratory, scientists can make DNA molecules from the mRNA found in a cell. This matching DNA is called cDNA (complementary DNA). If a biochip has all the genes of an organism dotted on its surface, then cDNA made from the mRNA in a cell can attach to (hybridize with) the genes on the chip. The more a gene is being expressed in the cell, the more cDNA for that gene there will be, and the more that cDNA will stick to the matching genes on the biochip. Spots with more labeled cDNA will glow more brightly under ultraviolet light. In this way, scientists can literally take a snapshot of how the genes in a cell are being expressed at any one time—how much the cell is making, at that moment, of thousands of different substances. This is extremely useful in trying to understand how cancer cells grow and in many other medical problems.

How complex cells arose on earth?

Prokaryotes were the first life on earth. There are fossil bacteria that are 3.8 billion years old, nearly as old as the oceans themselves. Being prokaryotes, these early cells did not have organized organelles enclosed by membranes. Prokaryotic life came on the scene very early in the earth’s history, but the next step took much longer. It was a billion years or more before complex cells with organelles, or eukaryotic cells, appeared. Strangely enough, this advance in cell design, shared by all multicellular life, probably started with a simple meal.

Biologists believe that mitochondria, the “powerhouses” of the cell, were once free-living bacteria. They are 1 to 3 microns (μm) long, about the same size as many bacteria. Like many bacteria, they contain layers of folded membranes. Although most of the cell’s DNA is contained in the nucleus, mitochondria have a small amount of their own DNA. This DNA is even in the form of a single, circular molecule like the chromosome of bacteria. When a cell needs new mitochondria it does not make them from scratch. Instead, the mitochondria within the cell divide in two, much as bacteria divide by cell division. Mitochondria resemble bacteria in several other respects.



Because of these similarities, biologists believe that mitochondria were originally bacteria that came to live inside other cells. The most likely scenario is that relatively large prokaryotic cells that ate smaller ones were unable to digest all of their food. It is also possible that the small cells were disease bacteria that invaded the larger cell but were unable to kill it. Both phenomena have been observed in the laboratory in living cells. Either way, the small cells eventually took up permanent residence in the larger cells, using them as hosts. The living together of different kinds of organisms like this is called symbiosis.

According to this theory, the host cells could not use oxygen in respiration. The smaller, symbiotic, bacteria did use oxygen and gave this ability to their hosts. This gave the host cells an advantage over cells that didn't have symbiotic bacteria.

Over the course of time, the host cells were able to transfer most of the symbiotic bacteria’s genes into their own nuclei, and thus came to control the symbionts. The bacteria and host cells became more and more dependent on each other. Eventually the bacteria became mitochondria.



Chloroplasts, the organelles that perform photosynthesis, are also thought to have arisen from symbiotic bacteria. Like mitochondria, they are about the right size, have a circular DNA molecule, and can reproduce themselves. Chloroplasts also contain folded membranes very similar to those of photosynthetic bacteria that we see today. In fact, biologists have found a possible “missing link” between photosynthetic bacteria and chloroplasts. Some sea squirts and other invertebrates contain symbiotic, photosynthetic bacteria called Prochloron that are similar to the chloroplasts of green algae and higher plants. It is thought that these chloroplasts arose from Prochloron-like bacteria when host cells ate the bacteria, some of which managed to resist being digested and became symbiotic.

The hypothesis that mitochondria and chloroplasts arose from bacteria, once highly controversial, is now almost universally accepted by biologists. Some biologists now even classify mitochondria and chloroplasts as symbiotic bacteria rather than organelles. Several other types of organelles may have similar origins. All multicellular organisms, including humans, carry in their very cells the legacy of these bacterial partnerships, probably the result of an ancient snack.

Complex cell

Tuesday, 20 May 2014

Use of Retroviral Vectors in Gene therapy

Some 24 per cent of all gene therapy clinical trials undertaken to date have employed retroviral vectors as gene delivery systems. Retroviruses are enveloped viruses. Their genome consists of ssRNA of approximately 8 kb. Upon entry into sensitive cells, the viral RNA is reverse transcribed and eventually yields double-stranded DNA. This subsequently integrates into the host cell genome. The basic retroviral genome contains a minimum of three structural genes : gag (codes for core viral protein), pol (codes for reverse transcriptase) and env (codes for the viral envelope proteins).

Retroviral life cycle


At either end of the viral genome are the long terminal repeats (LTRs), which harbour powerful promoter and enhancer regions and sequences required to promote integration into the host DNA. Also present, immediately adjacent to the 5' LTR, is the packing sequence (ψ). This is required to promote viral RNA packaging.

The ability of such retroviruses to (a) effectively enter various cell types and (b) integrate their genome into the host cell genome in a stable, long-term fashion, made them obvious potential vectors for gene therapy.

The construction of retroviruses to function as gene vectors entails replacing the endogenous viral genes, required for normal viral replication, with the exogenous gene of interest. Removal of the viral structural genes means that the resulting vector cannot itself replicate. 

Retroviruses display a number of properties/characteristics that influence their potential as vectors in gene therapy protocols. These may be summarized as follows:


  • Retroviruses as a group have been studied in detail and their biochemistry and molecular biology are well understood.


  • Most retroviruses can integrate their proviral DNA only into actively replicating cells.

  • The efficiency of gene transfer to most sensitive cell types is very high, often approaching 100 percent.

  • Integrated DNA can be subject to long-term, relatively high-level expression.

  • Proviral DNA integrates randomly into the host chromosomes.

  • Retroviruses are promiscuous, in that they infect a variety of dividing cell types.

  • Complete  copies of the proviral DNA are passed on to daughter cells if the original recipient cell divides.

  • Good, high-level, titre stocks of replication-incompetent retroviral particles can be produced.

  • Safety studies using retroviral vectors have already been carried out on various animal species.