MOLECULAR BIOLOGY
MOLECULAR BIOLOGY

Molecular biology; the study of biology at the molecular
level.

Molecular biology; the study of gene structure and
functions at the molecular level to understand the molecular
basis of hereditary, genetic variation, and the expression
patterns of genes.

The structure of DNA was described by British Scientists
Watson and Crick as long double helix shaped with its
sugar phosphate backbone on the outside and its bases on
inside; the two strand of helix run in opposite direction and
are anti-parallel to each other. The DNA double helix is
stabilized by hydrogen bonds between the bases.

This structure explains how genes engage in replication,
carrying information and acquiring mutation.
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The G+C content of a natural DNA can vary from 22-73%
and this can have a strong effect on the physical properties
of DNA, particularly its melting temperature.
General Structure of Nucleic Acid
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DNA and RNA are long chain
polymers of small compound
called nucleotides.
Each nucleotide is composed
of
a base;
sugar (ribose in RNA or
deoxyribose in DNA) and
a phosphate group.
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There are four different types of nucleotides found in DNA, differing only
in the nitrogenous base: A is for adenine; G is for guanine; C is for
cytosine and T is for thymine.
These bases are classified based on their chemical structures into two
groups: adenine and guanine are double ringed structure termed purine ,
thymine and cytosine are single ring structures termed pyrimidine.

The bases pair in a specific way:

Adenine A with Thymine T (two hydrogen bonds) and

Guanine G with Cytosine C (three hydrogen bonds).
Strands in the DNA runs antiparallel
FORM OF DNA
Characteristic
A-DNA
B-DNA
Z-DNA
2.6 nm
2.0 nm
1.8 nm
11
10.4
12
degrees rotation/bp
+32.7
+34.6
-30.0
axial distance/turn
2.8 nm
3.4 nm
4.5 nm
axial distance between bp
0.25 nm
0.33 nm
0.38 nm
diameter (D)
bp/turn
Replication proceeds in a semiconservative manner, each strand of the
helix serves as a template for the synthesis of complementary
strands. This lead to the formation of two complete copies of the
molecule, each consisting of one strand derived from the parent
molecule and one newly synthesized complementary strand.
DNA
DNA
DNA
DNA
GENOMIC DNA ORGANIZATION
Eukaryotic genes: DNA
molecules complexed with
other proteins especially
basic
proteins
called
histones,
to
form
a
substance
known
as
chromatin.
EUKARYOTIC CHROMATIN

Eukaryotic chromatin is
folded in several ways. The
first order of folding involves
structures called
nucleosomes, which have a
core of histones, around
which the DNA winds ( four
pairs of histones H2A,
H2B,H3 and H4 in a wedge
shaped disc, around it
wrapped a stretch of 147 bp
of DNA).
FUNCTION OF THE DNA

Deoxyribonucleic Acid (DNA), the gigantic molecule
which is used to encode genetic information for all life on Earth
excepts some viruses.

The chemical basis of hereditary and genetic variation are
related to DNA.

DNA directs the synthesis of RNA which in turn directs protein
synthesis.
Central Dogma of Molecular Biology
The flow of genetic information as follows:
MG331/MB331
DNA ISOLATION
OBJECTIVES

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To understand the basic process of isolation of DNA
from various sources eg blood, tissue, bacteria.
To realise that different types of DNA require different
methods of isolation.
To realise that the method used is dependent upon
the final application.
To understand the basis of gel electrophoresis
To realise that there are different types of gel
electrophoresis.
DNA EXTRACTION METHODS
Introduction
Deoxyribonucleic acid (DNA) isolation is an extraction process of
DNA from various sources. Methods used to isolate DNA are
dependent on the source, age, and size of the sample. Despite the
wide variety of methods used, there are some similarities among
them. In general, they aim to separate DNA present in the nucleus of
the cell from other cellular components.
Isolation of DNA is needed for genetic analysis, which is used for
scientific, medical, or forensic purposes. Scientists use DNA in a
number of applications, such as introduction of DNA into cells and
animals or plants, or for diagnostic purposes. In medicine the
latter application is the most common. On the other hand, forensic
science needs to recover DNA for identification of individuals (for
example rapists, petty thieves, accident, or war victims), paternity
determination, and plant or animal identification
Sources for DNA isolation are very diverse.
Basically it can be isolated from any living or
dead organism. Common sources for DNA
isolation include whole blood, hair, sperm,
bones, nails, tissues, blood stains, saliva,
buccal (cheek) swabs, epithelial cells, urine,
paper cards used for sample collection,
bacteria, animal tissues, or plants.
How Can We Recover DNA From a
Variety of Sources of Biological
Evidence?
Blood
Semen
Saliva
Urine
Hair (w/Root & Shaft)
Teeth
Bone
Tissue
Cigarette Butts
Envelope &
Stamps
Fingernail
Clippings
Chewing Gum
Bite Marks
Feces
WHAT ARE THE ESSENTIAL COMPONENTS OF A
DNA EXTRACTION PROCEDURE?
1.
2.
3.
4.
Maximize DNA recovery
Remove inhibitors
Remove or inhibit
nucleases
Maximize the quality of DNA
WHAT ARE THE MOST COMMONLY USED DNA
EXTRACTION PROCEDURES IN FORENSIC
SCIENCE?
Organic (Phenol-Chloroform) Extraction
 Non-Organic (Proteinase K and Salting out)
 Chelex (Ion Exchange Resin) Extraction
 FTA Paper (Collection, Storage, and Isolation)

The method utilized may be sample dependant,
technique dependant, or analyst preference
MG331/MB331
SPECIFIC METHODS OF DNA ISOLATION
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Genomic DNA
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Plasmid DNA
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Alkaline/SDS
Qiagen column methods
Bacteriophage M13 DNA
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SDS/Proteinase K
Qiagen columns
Alkaline method
Automated methods
PEG precipitaton method
Bacteriophage lambda DNA

PEG/Salt precipitation method
MG331/MB331
ISOLATION OF DNA
METHODS OF ISOLATING DNA
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Cell extraction
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Removal of cell debris
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proteins, lipids, polysaccharides
Concentration of DNA
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Organic - phenol, CHCl3
high salt
guanidinium HCl
ethanol, isopropranol
DNA absorbing matrix
CTAB, spermidine
Optional steps

Rnase A removal of RNA
ORGANIC EXTRACTION

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Perhaps the most basic of all procedures in forensic
molecular biology is the purification of DNA. The key
step, the removal of proteins, can often be carried out
simply by extracting aqueous solutions of nucleic acids
with phenol and/or chloroform.
Presence of proteins, lipids, polysaccharides and some
other organic or inorganic compounds in the DNA
preparation can interfere with DNA analysis methods,
especially with polymerase chain reaction (PCR). They
can also reduce the quality of DNA leading to its shorter
storage life
ORGANIC EXTRACTION
PROCEDURE
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Cell Lysis Buffer - lyse cell membrane, nuclei are
intact, pellet nuclei.
Resuspend nuclei, add Sodium Dodecly Sulfate
(SDS), Proteinase K. Lyse nuclear membrane
and digest protein.
DNA released into solution is extracted with
phenol-chloroform to remove proteinaceous
material.
DNA is precipitated from the aqueous layer by
the additional of ice cold 95% ethanol and salt
Precipitated DNA is washed with 70% ethanol,
dried under vacuum and resuspended in TE
buffer.
REAGENTS
Cell Lysis Buffer - Non-ionic detergent,
Salt, Buffer, EDTA designed to lyse outer
cell membrane of blood and epithelial
cells, but will not break down nuclear
membrane.
 EDTA (Ethylenediaminetetraacetic
disodium salt) is a chelating agent of
divalent cations such as Mg2+. Mg2+is a
cofactor for Dnase nucleases. If the
Mg2+is bound up by EDTA, nucleases are
inactivated.
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REAGENTS

Proteinase K - it is usual to remove most
of the protein by digesting with proteolytic
enzymes such as Pronase or proteinase K,
which are active against a broad
spectrum of native proteins, before
extracting with organic solvents.
Protienase K is approximately 10 fold
more active on denatured protein.
Proteins can be denatured by SDS or by
heat.
PURPOSE OF DNA EXTRACTION
To obtain DNA in a relatively purified
form which can be used for further
investigations, i.e. PCR, sequencing, etc
Overview of DNA Extraction
Break down
the cell wall
and
membranes
Centrifuge to
separate the
solids from
the dissolved
DNA
Dissolve
DNA
Precipitate
the DNA
using
isopropanol
Wash the
DNA pellet
with Ethanol
and dry the
pellet
Centrifuge to
separate the
DNA from
the dissolved
salts and
sugars
BASIC PROTOCOL

Most DNA extraction protocols consist of two parts
1.
A technique to lyse the cells gently and solubilize the DNA
2.
Enzymatic or chemical methods to remove contaminating proteins,
RNA, or macromolecules

In plants, the nucleus is protected within a nuclear membrane which is
surrounded by a cell membrane and a cell wall. Four steps are used to
remove and purify the DNA from the rest of the cell.
1. Lysis
2. Precipitation
3. Wash
4. Resuspension
LYSIS:
IN DNA EXTRACTION FROM PLANTS,
THIS STEP COMMONLY REFERS TO THE BREAKING
OF THE CELL WALL AND CELLULAR MEMBRANES (MOST
IMPORTANTLY, THE PLASMA AND NUCLEAR MEMBRANES)
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The cell wall (made of cellulose) is disrupted by mechanical
force (for example, grinding the leaves)
Then the addition of a detergent in the which breaks down the
cell membranes
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Detergents are able to disrupt membranes due to the amphipathic (having
both hydrophilic and hydrophobic regions) nature of both cellular
membranes and detergent molecules. The detergent molecules are able to
pull apart the membranes
The end result of LYSIS is that the contents of the plant cells
are distributed in solution.
PRECIPITATION :
THIS A SERIES OF STEPS WHERE DNA IS SEPARATED FROM
THE REST OF THE CELLULAR COMPONENTS
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In a research lab, the first part of precipitation uses phenol/chloroform to remove
the proteins from the DNA
 Phenol denatures proteins and dissolves denatured proteins.
 Chloroform is also a protein denaturant

The second part of research lab DNA precipitation is the addition of salts
 The salts interrupt the hydrogen bonds between the water and DNA
molecules.
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The DNA is then precipitated from the protein in a subsequent step with isopropanol
or ethanol
 In the presence of cations, ethanol induces a structural change in DNA
molecules that causes them to aggregate and precipitate out of solution.
The DNA is pelleted by spinning with a centrifuge and the supernatant removed.

Washing and Resuspension:
Washing:
The precipitated DNA is laden with acetate salts. It is
“washed” with a 70% ethanol solution to remove
salts and other water soluble impurities but not
resuspend the DNA.
Resuspension:
The clean DNA is now resuspended in a buffer to
ensure stability and long term storage.
The most commonly used buffer for resuspension is
called 1xTE
(The purpose of TE buffer is to solubilize DNA or RNA,
while protecting it from degradation) EDTA further
inactivates nucleases, by binding to metal cations
required by these enzymes
CHECKING THE QUALITY OF YOUR DNA
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The product of your DNA extraction will be used in
subsequent experiments
Poor quality DNA will not perform well in PCR
You will want to assess the quality of your DNA
extraction using the following simple protocol:
 Mix
10 µL of DNA with 10 µL of loading buffer
 Load this mixture into a 1% agarose gel
 Analyze results (the following slides provide guidance)
MG331/MB331
METHODS OF SEPARATING DNA
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Polyacrylamide gel electrophoresis
 20bp

- 2000bp
Conventional agarose gel electrophoresis
 300bp
- 40,000bp
 100bp-2000bp (special agaroses)
 low melting point agaroses
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Pulse field/CHEF
 40kbp
- 2000kbp
Analyzing DNA Samples
in a Research Lab
If properly done, genomic extraction should result in bright bands in
the very high base pair range of a gel electrophoresis.
Sizes of Genomic DNA for
various Species in kbp
E. Coli
Yeast
4,640,000bp
12,100,000bp
Fruit Fly 140,000,000bp
Human
3,000,000,000bp
Pea
4,800,000,000bp
Wheat
17,000,000,000bp
The genomic fragments run at ~12kbp because they are sheared during extraction
Nucleic Acid Analysis via UV Spectrophotometry
DNA Absorption Spectra
By measuring the amount of light absorbed by your sample at specific
wavelengths, it is possible to estimate the concentration of DNA and RNA.
Nucleic acids have an absorption peak at ~260nm.
[dsDNA] ≈ A260 x (50 µg/mL)
[ssDNA] ≈ A260 x (33 µg/mL)
[ssRNA] ≈ A260 x (40 µg/mL)
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