Download - Modern Biological Tools and Techniques

MODERN BIOLOGICAL

TOOLS AND TECHNIQUESPREPARED BY CHARLES PAUL BAUTISTA AND MA. FATIMA

CARINO

MODERN BIOLOGICAL TOOLS AND

TECHNIQUESMicroscope

Most popular tool in Biology. Used to

examine objects too small to be seen with

the naked eye.

Developed by Anton van Leeuwenhoek in

1670s.

The most common type is Compound

Microscope.


TECHNIQUES

Anton van Leeuwenhoek

Is known to have made over 500

microscopes, of which fewer than ten

have survived to the present day.


TECHNIQUESCompound Microscope

Commonly used in schools and is

equipped with lenses to enlarge objects

up to several times.

Used to examined cells and section of

tissues with the used of light to illuminate

an object being examined.


TECHNIQUES

Stereo Microscope

Used to examine the external structures of

a specimen, such as insects.


TECHNIQUES

Phase-contrast Microscope

Used to examine highly transparent

objects, such as unstained cells.


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Electron Microscope

Uses streams of electrons to enlarge

object up to 250 000 times.


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Transmission Electron Microscope

Is used to study internal structures of cells

through sectioned specimens.


TECHNIQUES

Scanning Electron Microscope

Is used to examine the surfaces or shapes

of objects, such as viruses.


TECHNIQUES

Fluorescent Microscope

Illuminates objects stained with

fluorescent dyes.

Had been used extensively in studying the

location of certain organelles or

substances inside the cell.


TECHNIQUES

Confocal Scanning Microscope

Used to examine the 3-dimensional

structure of a cell or organelles without

cutting the specimen into sections.


TECHNIQUES

Due to technological advancements in

image processing, objects examined using

the different microscopes can be

photographed or be viewed on TV or

computer screens. This process is calledvideo microscopy.

CELL AND TISSUE CULTUREThe use of cultured cells or tissues has an

advantage over using whole animals because of

ease of manipulation and simplicity of the system

without the complications of other cells or

substances in a whole organism.

Is achieved with the use of a medium containing

all the food requirements for a cell to survive grow

and multiply.

Is done under very strict, sterile(germ free)

conditions.

Is important in the production of monoclonal

antibodies called hybrid technology.

CENTRIFUGATIONCentrifuges

Are instruments used to separate cells or

cell organelles using centrifugal force.

Ordinary table-top centrifuges are used in

cell cultures to isolate whole cells from

culture media.

High-speed centrifuges or ultracentrifuges

are used to isolate different shapes and

sizes settle at the bottom of a centrifuge

tube at different sedimentation rates.

These are capable of spinning up to 75

000 revolutions per minute (rpm),

producing forces equivalent to around

500 000 times that of Earth’s gravity.

Chromatography

• It refers to a variety of techniques used

to purify biological molecules, such as

proteins and nucleic acids.

• A substance to be purified is suspended

in a liquid medium (mobile phase) and

is passed on to a column of matrix, such

as beads (immobile phase). The

substance to be purified interacts with

the matrix and the interaction is used as

a basis of separation.

Ion Exchange Chromatography

• Ionic charge of a substance is

used as the basis for purification.

Gel Filtration Chromatography

• Makes use of the size of the

molecule as the basis of

purification.

Affinity Chromatography

• Uses very special and very

specific interaction between

two molecules.

Gas Electrophoresis

is a powerful technique used to separate

and visualize proteins or nucleic acids.

Substances to be analyzed are driven

along a gel of cross- linked molecular

sieves using an electric current.

Substances to be analyzed are driven

along a gel of cross- linked molecular

sieves using an electric current.


TECHNIQUES

Sodium dodecyl sulfate-

polyacrylamide gel electrophoresis

(SDS-PAGE)

-It is therefore used to analyze the

molecular weight of a given protein.

-is used to analyze proteins based on

its molecular mass. It is therefore used

to analyze the molecular weight of a

given protein.

Isoelectric focusing

is a type of electrophoresis that

separates proteins according to

isoelectric pH.

Two-dimensional gel electrophoresis

An electrophoresis that combines

both SDS-PAGE and isoelectric

focusing

Agarose gel electrophoresis

is used to analyze and determine the

molecular weights of nucleic acids,

such as DNA or RNA.


TECHNIQUES

TYPES OF ELECTROPHORESIS

Spectrophotometry

Is an instrument used to determine the

concentration of proteins or nucleic

acids in a solution.

Used in bacterial cell cultures to estimate

the amount of cells present in a given

volume of cell culture medium.

Measure the amount of light at a

specific wavelength that is absorbed by

the solution, which is proportional to the

concentration of substances present in

the solution or the number of cells in a

medium.


TECHNIQUES

Polymerase Chain Reaction (PCR)

Is a method used to amplify or make copies of a given DNA fragment

using an enzyme called DNA polymerase.

Based on a principle that a double-stranded DNA molecule breaks into

two individual strands at high temperatures and with the use of PCR

primers (short DNA strands), the DNA polymerase can synthesize two

double-stranded DNA using two separated individual strands as

templates. When the process is repeated over and over again, there is

an exponential increase in the number of double- stranded DNA that is

produced: from one copy to two copies to four to eight to sixteen to thirty-two, and so on, depending on how many times the cycle is

repeated.

Widely used as a tool in DNA cloning, analysis of genetic diseases,

forensics, legal cases such as paternity testing and many more.


TECHNIQUES

DNA Sequencing

Is used to determine the sequence of nucleic

acids present in a given gene or DNA fragment.

This technology was independently developed

by Fredrick Sanger and Walter Gilbert.

Most automated DNA sequencers used today

are developed by Sanger.

Used to prepare the DNA sample to be

sequenced, followed by gel electrophoresis.

The results are tabulated and analyzed by a

computer.


TECHNIQUES

Immunoassays

Refer to a wide variety of techniques that use antibodies to

recognize a very specific substance called antigen, such as

protein.

Widely used in the development of diagnostic kits used in

hospitals to identify a particular disease or the presence of

bacterial and viral infections.

The pregnancy test kit is an example of an immunoassay

Western blot analysis

A type of immunoassay used to confirm the identity of a

protein immobilized into a membrane.

Enzyme-linked immunosorbent assay (ELISA)

Detects proteins or antibodies bound on a plastic plate (ELISA

plate) in a liquid system.

Immunofluorescence microscopy

Used to identify the location of certain organelles or proteins

inside a cell.


TECHNIQUES

DNA Cloning

Is a technique used to produce large quantities

of specific DNA fragments. The process involves

the ligation or linking of a gene of interest into a

cloning vector, such as bacterial plasmid, that

will serve as a carrier of the gene of interest.

Is used in a variety of applications including

gene analysis, DNA Sequencing, production of

recombinant proteins and industrial and medical

purpose, and the generation of transgenic

animals and plants.


TECHNIQUES

The fused gene and cloning vector called recombinant DNA, is then forced to enter a suitable host cell such as the bacterium E. coli.

The E. coli will then produce several copies of the recombinant DNA.

The recombinant DNA becomes a part of the E. coli cell and is then transmitted to the daughter cell every time the E. coli undergoes cell division.

Since E. coli divides at a very fast rate (~20 minutes), large amounts of the recombinant DNA can also be reproduced.

Microarrays (Gene Chips)

Is a relatively new technique used to identify genes

involved in a particular disease or genes involved in

the different processes inside the cell.

In this technique, thousands of genes are coated onto

DNA chips at precise microscopic locations using

robots.

Based on the principle of DNA hybridization (ability of

complementary strands of DNA to stick together),

DNA isolated from cells, such as those from patients,

are allowed to hybridize with the genes coated on the

array or DNA chip. By tagging the DNA of interest with fluorescent dyes, one can determine which gene it

bounds to. This technique has the advantage of

identifying several genes at the same time.


TECHNIQUES

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