Semeter-I M.Phil 2015 Zoology Department GCU Lahore

2. Presentation By : Sarwar Allah Ditta
Roll No # 657
Course Title: Advance Laboratory Techniques in Zoology
Course Code: Z-7236

3.  Vector: Types of vectors
 Plasmids: Isolation of plasmid
 Recombinant DNA
 Polymerase Chain Reaction
◦ Denaturing the DNA
◦ Annealing the primers to the DNA strands
◦ Extending the DNA Primers
 Genomic Library Construction
◦ Cosmid
◦ Bacterial Artificial Chromosomes (BAC)
◦ Yeast Artificial Chromosomes (YAC)
 Sub cloning using plasmid vectors
 Nucleic Acid Extraction
◦ Isolation of DNA
◦ Isolation of RNA
 Extraction of Amino Acids
 Extraction of proteins
 Extraction of Lipids
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4.  Molecular biology techniques are common methods
used in molecular biology, biochemistry, genetics and
biophysics which generally involve manipulation and
analysis of DNA, RNA, protein, and lipid.
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5. Plasmid is an extra-chromosomal DNA molecule separate from
the chromosomal DNA which is capable of replicating
independently of the chromosomal DNA.
Vector – a carrier (plasmid or other type) used for bringing
target DNA fragment into a host cell.
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6. Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kb
Bacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
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Types of Vector

7.  Cloning and sequencing of DNA and cDNA fragments
 Generation of genomic and cDNA libraries
 Expression of recombinant proteins
 Generation of mutant proteins
 Analysis of regulatory sequences
 Gene targeting
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8.  Origin of replication
 Antibiotic resistance
gene (Amp, Kan, Tet,
Chl)
 (Multiple cloning site)
Map of pOTB7 vector showing
Chloramphenicol resistance gene
(CMR), replication origin (ORI) and
multiple cloning site (MCS)
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9.  Multiple cloning site
 Promoter for cloned
sequence
 Reporter gene
 Tag
 Regulatory sequences
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10.  Replication origin defines the host bacteria: ColE1
replication origin is required for E.coli
 Replication origin may define the number of plasmid
copies per bacterial cell
 Bacteria may lose recombinant plasmid during cultivation
due to the absence of partitioning system (par). Naturally
occurring plasmids contain par that ensures that every
bacterial cell contains the plasmid.
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11.  Cut specific DNA sequence
 Protect bacteria from phage infection by digesting phage
DNA after injection
 Cellular DNA is protected by methylation that blocks
restriction enzyme activity
 Restriction enzyme (RE) means restricts virus replication
 Endonucleases are enzymes that produce internal cut called
as cleavage in DNA molecule
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12.  Presence of RE was postulated in 1960 by W.Arber
 The first true RE was isolated in 1970 by Smith, Nathans and
Arber. In 1978 they were awarded the Nobel Prize for
Phylsiology and Medicine.
 RE remain indispensible from molecular cloning and
sequencing.
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13. 13
 EcoRI Recognize hexameric sequence: 4-6
 Sau3A1 Recognize terameric sequence: 4-4
 Thus Sau3A1 cuts the same DNA molecule more
frequently
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14. 14
sticky ends
blunt ends
Recognition sequences and cut sites of various endonucleases
Restriction Digestion

15.  Creating genomic and cDNA libraries
 Cloning DNA molecules
 Studying nucleotide sequence
 Generating mutated proteins
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16. 1. Plasmid-containing bacteria are cultivated in
liquid media, supplemented with the
antibiotics for 18 h at 37°C with intensive
shaking
2. Cells are harvested by centrifugation
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Plasmid Preparation and Isolation

17. 3 solutions strategy
1. Resuspend in hypotonic buffer with RNase (buffer P1)
2. Lyse bacteria using NaOH/SDS solution (buffer P2)
3. Neutralize NaOH and precipitate proteins using NaAc
buffer (buffer P3)
Plasmid can be isolated from obtained lysate using
various strategies.
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Plasmid Preparation and Isolation

18. 1. Ethanol or Isopropanol precipitation
2. Silica matrix bind-wash-elute procedure
3. Density gradient centrifugation
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Plasmid Preparation and Isolation

19. 1. Ethanol is added to the lysate
2. Obtained sample incubated for 30 min
3. DNA is collected by centrifugation
• Cheap
• Fast
Advantages
Disadvantages
• Small amounts of DNA
• Poor purity, not sufficient for
applications like transfection and in
vitro translation
• Concentration of the plasmid can not
be determined photometrically
Also known as mini prep
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Plasmid Preparation and Isolation

20. 1. Apply lysate on the column
2. Wash the column
3. Elute the plasmid
4. Precipitate
• High purity of the plasmid
• Fast
Advantages Disadvantages
• Expensive
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Plasmid Preparation and Isolation

21. 1. Mix lysate with CsCl solution
2. Add EtBr
3. Centrifuge in the ultracentrifuge for 12-36m
4. Collect the plasmid
5. Precipitate
• The very best plasmid purity
• Relatively cheap
Advantages Disadvantages
• Slow
• Expensive equipment is needed
• High concentrations of EtBr
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Plasmid Preparation and Isolation

22. Photometric measurement of DNA concentration
UV 260 nm
Conc=50xOD260
Important! Photometric measurement of DNA concentration can not be
applied for “quick and dirty” plasmids, because of the presence of RNA
rests.
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Plasmid Preparation and Isolation

23. Gel electrophoresis is a technique used for the
separation of deoxyribonucleic acid (DNA),
ribonucleic acid (RNA), or protein molecules
using an electric current applied to a gel matrix.
Ethidium bromide stained agarose gel of
total RNA (1-3) and DNA ladder (M)
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Plasmid Preparation and Isolation
Selection of agarose concentration
Plasmid on an agarose gel

24. Recombinant DNA technology procedures by which
DNA from different species can be isolated, cut and
spliced together -- new "recombinant " molecules are
then multiplied in quantity in populations of rapidly
dividing cells (e.g. bacteria, yeast).
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25.  Restriction Enzyme
 Ligases
 Polymerase enzyme
 Host
 Vector
 Gene library
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26. 26

27. 27

28. • Recombinant DNA technology has a wide range of
application in industries, medical science, and
agriculture as "well as molecular biology.
 Molecular diagnosis of diseases
 Gene therapy
 DNA finger printing
 Production of vaccines
 Commercial and pharmaceutical products
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29. In this reaction, multiple copies of the gene of
interest is synthesised in vitro by using two
sets of primers and the enzyme DNA
polymerase.
1. Denaturation of ds DNA template
2. Annealing of primers
3. Extension of ds DNA molecules
Polymerase Chain Reaction
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30.  Temperature: 92-94C
 Double stranded DNA melts single stranded
DNA
92C
3’5’
3’ 5’
+
5’3’
5’ 3’
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Polymerase Chain Reaction

31.  Temperature: ~50-70C (dependant on the melting
temperature of the expected duplex)
 Primers bind to their complementary sequences
5’3’
5’ 3’
Forward primer Reverse primer
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Polymerase Chain Reaction

32.  Temperature: ~72C
 Time: 0.5-3min
 DNA polymerase binds to the annealed primers and
extends DNA at the 3’ end of the chain
Taq
5’
3’
Taq5’
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Polymerase Chain Reaction

33. Cycling
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Polymerase Chain Reaction

34.  DNA – 1 copy
 Known sequence Sequence of interest Known sequence
 PCR
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Polymerase Chain Reaction

35. 35Zoology Department GCU Lahore.
Polymerase Chain Reaction

36.  1) DNA sequence of target region must be known.
2) Primers - typically 20-30 bases in size.
These can be readily produced by commercial companies. Can also
be prepared using a DNA synthesizer
 3) Thermo-stable DNA polymerase - eg Taq polymerase which is
not inactivated by heating to 95C
4) DNA thermal cycler - machine which can be programmed to
carry out heating and cooling of samples over a number of cycles.
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Polymerase Chain Reaction

37. Molecular Identification Sequencing Genetic Engineering
Molecular Archaeology Bioinformatics Site-directed mutagenesis
Molecular Epidemiology Genomic Cloning Gene Expression Studies
Molecular Ecology Human Genome Project
DNA fingerprinting
Classification of organisms
Genotyping
Pre-natal diagnosis
Mutation screening
Drug discovery
Genetic matching
Detection of pathogens
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Polymerase Chain Reaction

38. 1. Breaking the cells open, commonly referred to as cell
disruption or cell lysis, to expose the DNA within. This is
commonly achieved by grinding, sonicating or treating
the sample with lysis buffer .
2. Removing membrane lipids by adding a detergent.
3. Removing proteins by adding a protease (optional
but almost always done).
4. Precipitating the DNA with an alcohol — usually ice-
cold ethanol or isopropanol. Since DNA is insoluble
in these alcohols, it will aggregate together, giving
a pellet upon centrifugation. This step also removes
alcohol-soluble salt.
Depending on the material and requirement the extraction
process can be modified with alteration of chemicals used
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39. Step 1: Disruption of cell walls by grinding
Step 2: Lysis of cells in extraction buffer
Step 1+2: mechanical disruption and
homogenization in extraction buffer
Extraction/Precipitation Method
Grind sample into a fine powder to
shear cell walls and membranes
Mix thoroughly with extraction
buffer to dissolve cell membranes
and inhibit nuclease activity
A homogenizer allows cells to be
mechanically disrupted within the
extraction buffer
Crude lysate
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40. Crude lysate containing
nucleic acids and other
cell constituents
Mix thoroughly with
an equal volume of
organic solvent
e.g. phenol, chloroform,
or phenol:chloroform
Centrifuge
The aqueous phase contains water-
soluble molecules, including nucleic
acids. Proteins and lipids become
trapped in the organic phase, and
are thus separated away. Insoluble
plant debris become trapped in the
interphase between the two layers
Perform additional extractions for increased purity
Collect aqueous phase
Extraction/Precipitation Method
Step 3: Organic extraction
Organic
Aqueous
Interphase
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41. • Pellet down nucleic acids.• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
After
Add alcohol and salt to
precipitate nucleic acids
from the aqueous fraction
Supernatant
Pellet
70% EtOH
Dissolve pellet
(H2O, TE, etc.)
Step 4: Nucleic Acid Precipitation
Extraction/Precipitation Method
Before After
Centrifuge Wash Centrifuge
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42. Each protein have unique amino acid
composition, sequence, subunit structure, size,
shape, net charge, isoelectric point, solubility,
heat-stability, hydrophobicity, ligand/metal
binding properties.
Proteins Extractions
SEPARATION METHODS:
•Precipitation
•Chromatography
•Centrifugation
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43.  Extraction
 Depending on the source, the protein has to be brought
into solution by breaking the tissue or cells containing it.
 There are several methods to achieve this:
 Repeated freezing and thawing,
 sonication,
 Homogenization by high pressure,
 filtration, or permeabilization by organic solvents.
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Proteins Extractions

44.  In bulk protein purification, a common first step to
isolate proteins is precipitation with ammonium
sulfate (NH4)2SO4.
 This is performed by adding increasing amounts of
ammonium sulfate and collecting the different
fractions of precipitate protein. Ammonium sulphate
can be removed by dialysis.
 The hydrophobic groups on the proteins gets exposed
to the atmosphere and it attracts other protein
hydrophobic groups and gets aggregated. Protein
precipitated will be large enough to be visible.
 One advantage of this method is that it can be
performed inexpensively with very large volumes.
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Proteins Extractions

45.  Usually a protein purification protocol contains one or
more chromatographic steps.
 The basic procedure in chromatography is to flow the
solution containing the protein through a column
packed with various materials.
 Usually proteins are detected as they are coming off
the column by their absorbance at 280 nm.
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Proteins Extractions

46.  Using porous matrix
 Based on different sizes of
proteins
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Proteins Extractions

47.  Anion exchange resins (positive
charge ) separate negatively charged
compounds
 cation exchange resins (negative
charge) separate positively charged
molecules
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Proteins Extractions

48.  Affinity Chromatography
is a separation technique
based upon molecular
conformation
 resins have ligands
attached to their surfaces
which are specific for the
compounds to be
separated.
 ligands function in a
fashion similar to that of
antibody-antigen
interactions.
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Proteins Extractions

49.  high pressure to drive the solutes
through the column faster.
 diffusion is limited and the resolution is
improved.
 The most common form is "reversed
phase" hplc, where the column material
is hydrophobic. The proteins are eluted
by a gradient of increasing amounts of
an organic solvent, such as acetonitrile.
The proteins elute according to their
hydrophobicity. After purification by
HPLC the protein is in a solution that
only contains volatile compounds, and
can easily be lyophilized
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Proteins Extractions

50. The lipid extraction by Folch method
Homogenized by using 2:1
chloroform/methanol mixture
Filltration by (fillter paper or
centrifuge)
Washing with (water or NaCl solution)
The mixture is centrifuge
Remove the upper phase (Methanol)
by siphoning
The lower phase (Chloroform)
Result: LIPIDS
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