Republic of the Philippines

Department of Education
Region III – Central Luzon
SCHOOLS DIVISION OF ZAMBALES
ZAMBALES NATIONAL HIGH SCHOOL
ZONE VI, IBA, ZAMBALES

Biotechnology 8
Quarter 1 Week 7

I. Learning Competency
Describe the Biological techniques, procedures and methods

BIOLOGICAL TOOLS AND TECHNIQUES

Microscope
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.

a. Compound 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.

b. Stereo Microscope
Used to examine the external structures of a specimen, such as insects.

c. Phase-contrast Microscope
Used to examine highly transparent objects, such as unstained cells.

d. Electron Microscope Uses streams of electrons to enlarge object up to

250 000 times.
e. Transmission Electron Microscope Is used to study internal
structures of cells through sectioned specimens.

f. Scanning Electron Microscope Is used to examine the surfaces or

shapes of objects, such as viruses.

g. Fluorescent Microscope
Illuminates objects stained with fluorescent dyes.
Had been used extensively in studying the location of
certain organelles or substances inside the cell.

h. Confocal Scanning Microscope

Used to examine the 3-dimensional structure of a cell or organelles
without cutting the specimen into sections.
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 called video microscopy.

CELL AND TISSUE CULTURE

The 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.

CENTRIFUGATION
Centrifuges
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 can spin
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.

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

Video of Gel Electrophoresis

https://www.youtube.com/watch?v=ZDZUAleWX78

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.

Types of Electrophoresis
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.

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.

Polymerase Chain Reaction (PCR)

• Developed by Mullis, who was awarded the Noble Prize in 1992

• PCR is a process that allows the production of many copies of DNA fragments
• PCR uses heat (temperatures between 94 º C -96 º C) to separate the 2 strands of DNA by breaking the H-bonds between
complimentary base pairs

The two strands are used for replication

• Temperature is lowered to 50 º C- 60 º C and DNA primers attach to the DNA templates.
• At 72 º C Taq polymerase identify the DNA primers and begin to attach free nucleotides to complete the
complimentary strand of each template.
• When the replication is complete, the process is repeated.

Since each new cycle has more templates that can be used for replication, each cycle results in an “exponential increase
in the number of copies of the target DNA”

This process can result in 2 types of strands: Variable-length strands: A mixture of strands of DNA that have been
replicated and are of unequal length Constant length strands: A mixture of strands of DNA that have been replicated and
are of equal length

Uses of PCR:
• Forensic criminal investigations: a single cell can be replicated many times, therefore only a small sample of DNA
evidence is required from a crime scene
• Medical Diagnosis: to detect the presence of HIV
• Genetic Research uses fossil remains to determine if 2 species are closely related

 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.

Video of PCR: http://www.youtube.com/watch?v=_YgXcJ4n-kQ&feature=related

DNA Sequencing
Is used to determine the sequence of nucleic acids present in each 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.

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 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

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.
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 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, can 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.

• 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
• 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

References
https://www.slideshare.net/Fatima_Carino23/modern-biological-tools-and-techniques
https://www2.slideshare.net/ihmcbiology1213/prep-and-modern-bio-techniques
https://www.slideserve.com/ellard/advanced-molecular-biological-techniques