分子生物学

"... not so much a technique as an approach, an approach from the viewpoint of the so-called basic sciences with the leading idea of searching below the large-scale manifestations of classical biology for the corresponding molecular plan. It is concerned particularly with the forms of biological molecules and ..... is predominantly three-dimensional and structural - which does not mean, however, that it is merely a refinement of morphology - it must at the same time inquire into genesis and function"

[编辑] 同其他 "分子水平" 生物科学的关系

Schematic relationship between biochemistry, genetics and molecular biology

分子生物学的研究者们不仅适用分子生物学特有的技巧(see Techniques section later in article), 而且越来越多地从基因学,生物化学和生物物理学的技巧和思路中获得启迪，综合利用。从而这些学科间不再存在一条硬性的分割线，如果以前曾经有过的话。下图抽象地展示了这些领域之间相互关系的一种描述:

Biochemistry is the study of the chemical substances and vital processes occurring in living organisms.
Genetics is the study of the effect of genetic differences on organisms. Often this can be inferred by the absence of a normal component (e.g. one gene). The study of "mutants" – organisms which lack one or more functional components with respect to the so-called "wild type" or normal phenotype. Genetic interactions such as epistasis can often confound simple interpretations of such "knock-out" studies.
Molecular biology is the study of molecular underpinnings of the process of replication, transcription and translation of the genetic material. The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, despite being an oversimplified picture of molecular biology, still provides a good starting point for understanding the field. This picture, however, is undergoing revision in light of emerging novel roles for RNA.

Much of the work in molecular biology is quantitative, and recently much work has been done at the interface of molecular biology and computer science in bioinformatics and computational biology. As of the early 2000s, the study of gene structure and function, molecular genetics, has been amongst the most prominent sub-field of molecular biology.

Increasingly many other fields of biology focus on molecules, either directly studying their interactions in their own right such as in cell biology and developmental biology, or indirectly, where the techniques of molecular biology are used to infer historical attributes of populations or species, as in fields in evolutionary biology such as population genetics and phylogenetics. There is also a long tradition of studying biomolecules "from the ground up" in biophysics.

[编辑] Techniques of molecular biology

Since the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells.

[编辑] Expression cloning

One of the most basic techniques of molecular biology to study protein function is expression cloning. In this technique, DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes) into a plasmid (known as an expression vector). This plasmid may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid.

This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells is called transformation, and can be completed with several methods, including electroporation, microinjection, passive uptake and conjugation. Introducing DNA into eukaryotic cells, such as animal cells, is called transfection. Several different transfection techniques are available, including calcium phosphate transfection, liposome transfection, and proprietary transfection reagents such as Fugene. DNA can also be introduced into cells using viruses or pathenogenic bacteria as carriers. In such cases, the technique is called viral/bacterial transduction, and the cells are said to be transduced.

In either case, DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.

[编辑] Polymerase chain reaction (PCR)

主條目：Polymerase chain reaction

The polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases of DNA. PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library.

[编辑] Gel electrophoresis

主條目：Gel electrophoresis

Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is that DNA, RNA, and proteins can all be separated using an electric field. In agarose gel electrophoresis, DNA and RNA can be separated based on size by running the DNA through an agarose gel. Proteins can be separated based on size using an SDS-PAGE gel. Proteins can also be separated based on their electric charge, using what is known as an isoelectric gel.

[编辑] Southern blotting

主條目：Southern blot

Named after its inventor, biologist Edwin Southern, the Southern blot is method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capilliary action. The membrane can then be probed using a DNA probe labeled using a complement of the sequence of interest. Most original protocols used radioactive labels, however now non-radioactive alternatives are available. Southern blotting is less commonly used in laboratory science due to the capacity of using PCR to detect specific DNA sequences from DNA samples. However, these blots are still used for some applications, such as measuring transgene copy number in transgenic mice, or in the engineering of gene knockout embryonic stem cell lines.

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[编辑] Northern blotting

主條目：Northern blot

The Northern blot is used to study the expression patterns a specific type of RNA molecule as relative comparison among of a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used, however, most result in the revelation of bands representing the sized of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expressing is occurring by measuring how much of that RNA is present in different samples. It is one of the most basic tools for determining at what time certain genes are expressed in living tissues.

[编辑] Western Blotting

主條目：Western blot

Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey ([[polyclonal antibody|polyclonal antibodies)or produced in cell culture (monoclonal antibodies). These antibodies can be used for a variety of analytical and preprative techniques.

In Western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE (for Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including coloured products, chemiluminescence, or autoradiography.

Analogous methods to western blotting can also be used to directly stain specific proteins in cells and tissue sections. However, these immunostaining methods are typically more associated with cell biology than molecular biology.

The terms "Western" and "Northern" are jokes: The first blots were with DNA, and since they were done by Ed Southern, they came to be known as Southerns. I don't think Patricia Thomas, inventor of the RNA blot, which became known as a "Northern", actually used the term. ^[2]. To carry the joke further, one can find reference in the literature [[1]] to "southwesterns" (Protein-DNA interactions) and "farwesterns" (Protein-Protein interactions).

[编辑] Abandoned Technology

主條目：Older Technology

As new procedures and technology become available, the older technology is rapidly abandoned. A good example is methods for determining the size of DNA molecules. Prior to gel electrophoresis, with agarose and polyacrylamide, DNA was sized with rate sedimentation in sucrose gradients, a slow and labor intensive technology requiring expensive instrumentation; prior to sucrose gradients, viscometry was used. Aside from their historical interest, it is worth knowing about older technology as it may be useful to solve a particular problem.

[编辑] History

主條目：History of molecular biology

Molecular biology was established in the 1930s, the term was first coined by Warren Weaver in 1938 however. Warren was director of Natural Sciences for the Rockefeller Foundation at the time and believed that biology was about to undergo a period of significant change given recent advances in fields such as X-ray crystallography. He therefore channeled significant amounts of (Rockefeller Institute) money into biological fields.

[编辑] Further reading

Keith Roberts, Martin Raff, Bruce Alberts, Peter Walter, Julian Lewis and Alexander Johnson, Molecular Biology of the Cell

4th Edition, Routledge, March, 2002, hardcover, 1616 pages, 7.6 pounds, ISBN 0-8153-3218-1

3th Edition, Garland, 1994, ISBN 0-8153-1620-8

2nd Edition, Garland, 1989, ISBN 0-8240-3695-6

[编辑] Notes

[编辑] See also

Cell biology (structures and components of the cell)
DNA and chromosome structure
Protein biosynthesis (transcription from DNA to RNA, translation from RNA into protein)
Protein structure and diversity
Genome
Important publications in molecular biology
List of molecular biology topics
Proteome

[编辑] Notable molecular biologists

Francis Crick
James D. Watson
Maurice Wilkins
Erwin Chargaff
Rosalind Franklin
Max Perutz
Susumu Tonegawa
Christiane Nüsslein-Volhard
Frederick Sanger
Francois Jacob

[编辑] In fiction and games

Genome soldiers (MGS)

[编辑] External links

Frederick Sanger Freeview Video Interview/Documentary by the Vega Science Trust.
Max Perutz Freeview Video interview with Max Perutz by the Vega Science Trust.
Christiane Nüsslein-Volhard Freeview interview by the Vega Science Trust.
The Collection of Biostatistics Research Archive
Statistical Applications in Genetics and Molecular Biology
The International Journal of Biostatistics
The International Journal of Biological Sciences
Institute of Molecular and Cell Biology
Nature Reviews Molecular Cell Biology (journal home)
Stanford Encyclopedia of Philosophy entry
The Virtual Library of Biochemistry and Cell Biology
A brief history of molecular biology
A Monk's Flourishing Garden: the Basics of Molecular Biology Explained - a review from the Science Creative Quarterly
The Molecular Biology Gateway
Scientific American Magazine (April 2004 Issue) Evolution Encoded
National Center for Biotechnology Information

[编辑] References

Cohen, S.N., Chang, A.C.Y., Boyer, H. & Heling, R.B. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA 70, 3240 – 3244 (1973).
Rodgers, M. The Pandora's box congress. Rolling Stone 189, 37 – 77 (1975).

↑ W.T. Astbury, [Nature 190, 1124 (1961)]
↑ Patricia S. Thomas, Hybridization of Denatured RNA and Small DNA Fragments Transferred to Nitrocellulose PNAS 1980; 77: 5201-5205 www.pnas.org

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