Preparation Techniquesand

Modern Biological Techniques

• Cell and Tissue Culture• Centrifugation• Chromatography• Gel Electrophoresis• Spectrophotometry• Polymerase Chain Reaction• DNA Sequencing• Immunoassays• DNA Cloning• Microarrays

Cell and Tissue Culture

• Is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment.

• Culture cells derived from multi-cellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes, including viruses, bacteria and protists.

• Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and other products of biotechnology i.e. drug discovery, cancer biology, regenerative medicine and basic life science research

• Tissue culture is the growth of tissues or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar.

• Tissue culture commonly refers to the culture of animal cells and tissues,

Centrifugation• The process of separating lighter portions of a solution, mixture, or

suspension from the heavier portions by centrifugal force.• A laboratory centrifuge is a piece of laboratory equipment, driven by a motor,

which spins liquid samples at high speed.– Where the centripetal acceleration is used to separate substances of

greater and lesser density.• • There are various types of centrifugation:• Differential centrifugation, often used to separate certain organelles from

whole cells for further analysis of specific parts of cells• Isopycnic centrifugation, often used to isolate nucleic acids such as DNA• Sucrose gradient centrifugation, often used to purify enveloped viruses and

ribosomes, and also to separate cell organelles from crude cellular extracts

Chromatography

• Is the collective term for a set of laboratory techniques for the separation of mixtures.

• The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.

• The various constituents of the mixture travel at different speeds, causing them to separate.

Gel Electrophoresis• Involves the separation of chemicals along a solid medium in the presence of an

applied potential difference. • Chemicals such as blood proteins, DNA or inorganic ions can be separated

according to differences in their mass and/or charge. The solid medium used in electrophoresis is usually an agarose or polyacrylamide gel

• Has uses in forensic science because it can be used to isolate and compare DNA, blood proteins and inorganic substances such as gunshot residues from crime scenes with suspects, victims or standard reference material.– produce DNA fingerprints; DNA evidence from a crime scene can be compared

to DNA samples from different suspects, for instance, and suspects can either be included or excluded from suspicion using the results of such tests

Spectrophotometry

• A method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution.

• The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical substance.

• One of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications.

Polymerase Chain Reaction• The polymerase chain reaction (PCR) is a biochemical technology in

molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

• Now a common and often indispensable technique used in medical and biological research labs – i.e. DNA cloning for sequencing, – functional analysis of genes; – diagnosis of hereditary diseases; – identification of genetic fingerprints (used in forensic sciences and

paternity testing); – detection and diagnosis of infectious diseases.

DNA Sequencing• Includes several methods and technologies that are used for determining

the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.

• Knowledge of DNA sequences has become indispensable for basic biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as diagnostic, biotechnology, forensic biology and biological systematics.

• Instrumental in the sequencing of the human genome, in the Human Genome Project.

• Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genomes.

Immunoassays• A specific type of biochemical test that measures the presence or concentration of a

substance (referred to as the "analyte") in solutions that frequently contain a complex mixture of substances.

• Analytes in biological liquids such as serum or urine are frequently assayed (i.e., measured) using immunoassay methods.

• In essence, the method depends upon the fact that the analyte in question is known to undergo a unique immune reaction with a second substance, which is used to determine the presence and amount of the analyte.

• This type of reaction involves the binding of one type of molecule, the antigen, with a second type, the antibody. Immunoassays can be carried out using either the antigen or the antibody in order to test for the other member of the antigen/antibody pair. In other words, the analyte may be either the antigen or the antibody.

• Applied in measurement of blood levels of vitamins, hormones, etc. Also used in sports anti-doping laboratories to test blood samples for prohibited human growth hormone.

• .

DNA Cloning

• The use of DNA manipulation procedures to produce multiple copies of a single gene or segment of DNA.

Microarrays

• A small solid support, usually a membrane or glass slide, on which sequences of DNA are fixed in an orderly arrangement.

• Used for rapid surveys of the expression of many genes simultaneously, as the sequences contained on a single microarray can number in the thousands.

• Fixation – cutting of material into relatively tiny pieces and soaking it in a fixative

• Staining – allows one to observe clearly structural details of the specimen to be observed- provides contrast through color that reveals structural details undetected in other preparations- ex. of stains: iodine, methylene blue

• Computerized Axial Tomography (CAT Scan) – uses a rotating beam of x-rays. Provides a detailed picture of a human body

• Chromatography – separating and analyzing a mixture of chemical substances

• Tissue Culture – growing living cells from organisms in culture tubes filled with oxygen and all necessary nutrients

• Centrifugation – separating materials of different densities

• Mounting – prepare specimen for microscopic examination, especially by positioning on a slide

• Dry Mount – most basic; position specimen on slide and put on cover slip- Dry Mount samples: hair, feather, pollen

• Wet Mount – suspend specimens in fluids like water, immersion oil, etc.- Wet Mount samples: aquatic samples, living organisms, natural observations

• Smearing – the sample is spread on a slide• Squashing – spreads specimens by pressure

• Fixative Agent – substance used for preservation of tissue/cell specimens for later examination

• Microdissection – dissection under magnification

• Dehydration – extracting water from tissues/cells through techniques like heating or by using dehydrating agents like alcohols