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Chemical Analysis

indicator method

a simple test determine simple chemical


reactions via cellular components
ex. Litmus test, phenol test (acid amino
group, base hydroxyl group)
it indicates color changes because of affinity of
Hydrogen and hydroxyl group
involves:
presence of residue/precipitate
solubility
gas evaluation test
indirect chemical test
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Presence of
Residue/Precipitate

it means there is
chemical bonding
basically produce
precipitate like
crystalline, coagulates,
granules

Solubility
use in materials that are
typically immiscible (cannot
mix each other ) or low
solubility
layer formation (lipids)

Gas Evaluation Test

test of chemical
wherein there is a
formation of active
gas liberation
through bubble
formation called

Indirect Chemical test

does not allow you to


determine the exact
component unless
subject the test to
thorough chemical test
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BIOCHEMICAL TEST
identify the main important
chemical compounds
each test take a small amount of
the substance to test, shake it in
water in a test tube( the substance
may need grinding with a pestle
and mortar, to break up the cells
and release the cell contents
many of these compounds are

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BIOCHEMICAL TEST

benedicts rest for


reducing sugar
Benedicts test for nonreducing sugar
Iodine test for starch
Emulsion test for lipids

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Benedicts Rest for


Reducing Sugar
all monossacharides and
most dissacharides will
reduce copper (II) sulphate
producing a precipitate of
copper (I) oxide on heating,
so they are called reducing
sugars
Benedicts reagent is an

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Benedicts Rest for


Reducing Sugar
grind up sample
to aproximate 2cm3 of test
solution add equal quantity of
Benedicts reagent
shake, and heat for a few
minutes at 95 C in a water
bath
a precipitate indicates

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Benedicts Test for Nonreducing Sugar


non-reducing sugars do not
reduce copper sulphate.
However, if it is first
hydrolysed to its constituent
monossacharides, it will then
give a positive Benedicts test
first test a sample for
reducing sugars, to see if

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Benedicts Test for Nonreducing Sugar


Using a separate sample;
boil the test solution with dilute
hydrochloric acid for a few minutes to
hydrolyse the glycosidic bond
neutralize by adding small amounts of
solid sodium hydrogen carbonate until
it stops fizzing
perform the Benedicts test
a positive result indicates the presence
of simple non-reducing sugar
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Iodine Test for Starch


to approximately 2cm of the
test solution add 2 drops of
iodine/potassium iodide
solution
a blue/black color indicates
the presence of starch
starch is the only slightly
soluble in water, but the test

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Emulsion Test for Lipids

lipids do not dissolve in water, but do


dissolve in ethanol. This characteristic
is used in the emulsion test
grind up sample
shake some test sample with about
4cm3 of ethanol
decant the liquid into a test tube of
water leaving any un-dissolved
substances between
if there are lipids dissolved in the
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Biuret Test for Proteins

to about 2cm3 of test solution add an


equal volume of biuret solution, down
the side of the test tube
a blue ring forms at the surface of the
solution, which disappears on shaking
and the solution turns lilac-purple
indicating protein
the color change is due to a complex
between nitrogen atoms in a peptide
chain and copper (II) ions, so this is
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1.

Tools used in Molecular


Biology
Gel Electrophoresis
a.

b.

c.

The basic principles is that DNA,


RNA and proteins can all be
separated by means of an electrical
field
In agarose gel electrophoresis, DNA
and RNA can be separated on the
basis of size by running the DNA
through an agarose gel
Proteins can be separated on the
basis of size by using an SDS-PAGE
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Tools used in Molecular


Biology
2. Polymerase Chain Reaction (PCR)
a. is an extremely versatile techniques
for copying DNA
b. PCR allows a single DNA sequence to
be copied (millions of times) or altered
in predetermined ways
c. it has many variations, like reverse
transcription PCR (RT-PCR) for
amplification of RNA, and real-time PCR
(QPCR) which allow for quantitative
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Primer

is a stand of nucleic acid that serves as a


starting point for DNA synthesis
usually short, chemically synthesized
oligonucleotides, with a length of about
twenty bases. They are hybridized to a
target DNA, which is then copied by the
polymerase
minimum primer length used in most
applications is 18 nucleotides
replication starts at the 3-end of the
primer, and copies the opposite 42strand

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Application of PCR

is the study of patterns of gene


expression
the task of DNA sequencing can also
be assisted by PCR
PCR has numerous applications to the
more traditional process of DNA
cloning
phylogenic analysis of DNA from
ancient sources
a common application of PCR is the
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Macromolecule blotting and


probing
Southern Blotting

southern blot is a method for probing


for the presence of a specific DNA
sequence within a DNA sample
DNA samples are separated by gel
electrophoresis and then transferred
to a membrane by blotting via
capillary action
the membrane is then exposed to a
labeled DNA probe that has a
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Southern
Blotting

less commonly used due to


the capacity of other
techniques, such as PCR
still used for some
applications such as
measuring transgene copy
number in transgenic mice, or
in the engineering of gene
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Northern Blotting

used to study the expression


patterns of a specific type of
RNA molecule as relative
comparison among a set of
different samples of RNA

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Genetic markers
are sequences of DNA which have
been traced to specific locations on
the chromosomes and associated
with particular traits
it can be described as a variation
that can be observed
may be a short DNA sequence,
such as a sequence surrounding a
single-base pair change (single

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Some commonly used type of genetic


markers:
RFLP (Restriction fragment length
polymorphism)
AFLP (Amplified fragment length
polymorphism)
RAPD (Random amplification of
polymorphic DNA)
VNTR (Variable number tandem
repeat)
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Some commonly used type of


genetic markers:
SNP (Single nucleotide
polymorphism)
STR (Short tandem repeat)
SFP (Single feature
polymorphism)
DArT (Diversity arrays
technology)
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5 conditions of a suitable
DNA markers:
must be polymorphic
co-dominant inheritance
randomly and frequently
distributed throughout the
genome
easy and cheap to detect
reproducible

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Molecular markers
applications includes :
germplasm characterization
genetic diagnostics
Characterization of transformants
Study of genome
organization and phylogenetic
analysis
paternity testing and the
investigation of crimes

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RFLP
Restriction

Fragment length
polymorphsim involves
fragmenting a sample of DNA by
a restriction enzyme, which can
recognize and cut DNA wherever
a specific short sequence occurs
occurs when the length of a
detected fragment varies
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RFLP
advantages:
variant are co dominant
measure variation at the
level of DNA sequence, not
protein sequence

disadvantage:

requires relatively large


amount of DNA
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AFLP
Amplified fragment length
polymorphsim in this analysis we
can amplify restricted fragments
and reduces the complexity of
material to be analyzed (approx
1000 folds)
it can be used for comparison by
which closely related species only

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AFLP
Advantages includes:

fast
relatively inexpensive
highly variable

Disadvantage:
markers are dominant
presence of a band could mean
the individual is either
homozygous or heterozygous for
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RAPD
Random amplification of polymorphic
DNA is a type of PCR reaction, but the
segments of DNA that are amplified are
random
Advantages:
Fast - relatively inexpensive
- highly
variable
Disadvantage:
Markers are dominant
presence of a band could mean the

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Micro satellite
polymorphism, SSR or
Simple sequence repeat

or Short tandem Repeats (STRs) are


repeating sequences of 1-6 base pairs of
DNA
Advantages:
Highly variable
- fast evolving -co
dominant

Disadvantage:
Relatively expensive and time consuming
to develop
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SNP
A

single-nucleotide
polymorphism (SNP) is a
DNA
sequence
variation
occuring when a single
nucleotide A, T, C or G- in
the genome (or other shared
sequence differs between
members of a species or
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STR

short tandem repeat in DNA occurs


when a pattern of two or more
nucleotides are repeated and the
repeated sequences are directly
adjacent to each other
the pattern can range in length from 2
to 16 pairs (bp) (for ex. CATG) in a
genomic region) and is typically in the
non-coding intron region
used in forensic cases
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Principles of DNA
Isolation and Purification
DNA cam be isolated from any
nucleated cell
DNA is a giant anion in solution

Sources of DNA
- blood - Cultured cells (plant or
animal)
Bacteria - biopsies
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DNA isolation
is a routine procedure to
collect DNA for
subsequent molecular
analysis.
3 basic steps in DNA
extraction:
1. Cell disruption

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3 Basic steps in DNA


extraction:
1.

2.

3.

Cell disruption this is commonly


achieved by grinding or sonicating the
sample. Removing membrane lipids by
adding a detergent
Isolation of DNA- removing a
proteins by adding a protenease
(optional but almost always done)
Precipitating the DNA- usually icecold ethanol or isopropanol is used.
Since DNA is insoluble in these
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Basic Rules
blood first lyse (explode) the red
blood cells with a gentle detergent
such as Triton -X-100
Wash cell hemoglobin(and other
pigments) inhibits restriction
enzyme and TAQ polymerase
wear gloves to protect your
samples from you
autoclave all solutions and store in
fridge (except SDS and organic

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Getting the DNA:

Lyse all cells in presence of:


NaCl so that DNA is stabilised and remains
as a double helix
EDTA which chelates Mg++ and is a cofactor of DNAase which chews up DNA
rapidly
anionic detergent SDS which disrupts the
lipid bilayers, help to dissolve membranes
and binds positive charges of chromosant
proteins (histones) to release the DNA into
the solution
include a protease to digest the proteins
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incubate the solution at an elevated