Gene & Cell Technology

ABS-831

Course Introduction

What is a cell?
How many different types
of CELLS are there in a
human body?

Structure of a cell
Prokaryotic cell

Eukaryotic cell

An Idealized Animal Cell

About 300 different

types of CELLS are
there in a human body

Gene
What is Gene?
How many Genes are there in a human cell?
25,000-35,000

What is a technique?
A technique is a procedure used to accomplish a
specific activity or task:

Technology is the study of or a collection of

techniques
Scientific technique, any systematic method to
obtain information of a scientific nature

Gene & Cell Technology

Gene technology is the term given to a range of activities
concerned with understanding the expression of genes, taking
advantage of natural genetic variation, modifying genes and
transferring genes to new hosts

It is a tool that offers enormous benefits for human health,

disease prevention, food security and sustainability
Cell technology refers to the manipulation of cells for various
purpose including
Cell therapy
Production of biopharmaceuticals
Industrial products like enzymes

Molecular Biology Methods

Introduction
Purification of nucleic acids
Manipulation of Nucleic Acids in vitro
DNA restriction and ligation
Restriction mapping
DNA Modification
Synthesis and degradation of nucleic acids
Nucleic acid amplification
Polymerase chain reaction (PCR)
Identification of specific sequences
Analysis of nucleic acids
Gel Electrophoresis
Hybridization
Sequencing
Introduction to cloning
Genetic Manipulation in Prokaryotes (E. coli)
Cloning vectors in prokaryotes
Plasmid vectors
Viral vectors (lambda phage derivatives)
Cosmids
M13 derivatives

Introduction of genetic information in bacteria

Transformation and Transfection
Detection and identification of a clone
Selection of recombinants
Screening of clones
Genomic libraries, cDNA libraries
Genetic Manipulation in Eukaryotes
Genetic manipulation in yeasts
Eukaryotic expression vectors
Gene expression analysis
Purification of proteins
Proteins analysis using ELIZA
Electrophoresis
Hybridization (Immunoblotting)
Cellular techniques
Bacterial culture
In-vitro cell culture
Analysis of cells
Microscopy
FACS

Marks distribution

OHTs

35%

Quizzes

10%

Assignment

5%

Terminal exam

50%

Recommended Books:
1. Genetic Engineering by D.S.T. Nicholl
2. Gene cloning and manipulation by Christopher Howe
3. Diagnostic Techniques in Genetics by Jean-Louis Serre
4. Biotechnology: principles and applications by David P. Clark
& Nanette J. Pazdernik

Isolation/Purification of Nucleic Acids:

Basic Concepts and Principles

What is DNA?

 DNA determines the characteristics of all living

organisms.
DNA is composed of a four-letter nucleotide/molecule
alphabet referred to as A, T, C, and G.
The order of the alphabet determines the
characteristics of the living organism, much like the
order of letters in our alphabet determines the
words.

Each cell in the human body contains >3 BILLION

letters.

DNA
A polymer of deoxyribonucleotides
Double-stranded
Right handed helix
Individual deoxynucleoside triphosphates are coupled by
phosphodiester bonds
esterification
link 3 carbon of one ribose with 5 C of another
terminal ends : 5 and 3

a double helical structure

common axis for both helices
antiparallel relationship between 2 DNA strands

PERIPHERY OF DNA
SUGAR-PHOSPHATE CHAINS

CORE OF DNA
BASES ARE STACKED IN PARALLEL FASHION
CHARGAFFS RULES
A=T
G=C

COMPLEMENTARY BASE-PAIRING

Forces That Stabilize Nucleic Acid

Structures

SUGAR-PHOSPHATE CHAIN CONFORMATIONS

BASE PAIRING
BASE-STACKING, HYDROPHOBIC
IONIC INTERACTIONS

TYPES OF DNA:
Genomic (chromosomal)
Organellar (satellite)
Plasmid (extrachromosomal)
Phage/viral (ds or ss)
Complementary (mRNA)

The only difference between living

organisms is the amount and order of

the DNA alphabet.

RNA
Unlike DNA, RNA is synthesized as a single strand
There are double-stranded RNA structures
RNA can fold back on itself
Depends on base sequence

Gives stem (double-strand) and loop (single-strand

structures)

Are there any RNA-DNA hybrids in cells?

Hybrid DNA-RNA Structures

Usually short sequences

Examples:
DNA synthesis is initiated by RNA primers
DNA is the template for transcription to RNA

Why isolate DNA?

Isolation of DNA is often the first step before
further analysis DNA profiling (Forensic)
Cloning
Disease diagnosis
DNA sequencing

Genetically modified organisms (GMO), agriculture,

pharmaceutical
Environmental testing, bioterrorism

Structure of the cell

Plasma membrane and membranes of
organelles (nuclear envelope included)
DNA located in nucleus
A lot of proteins around
Mitochondrial DNA

Isolation of Nucleic Acids

Goals:
Removal of proteins
DNA vs RNA
Isolate specific type of nucleic acid

Steps to DNA Extraction

1. Break the cells open to expose DNA (cell lysis)
Chemical & physical methods (blending, grinding, sonicating
the samples)

2. Remove membrane lipids by adding detergent or

surfactants
3. Removing proteins by adding protease (optional)
4. Removing RNA by adding Rnase
5. DNA purification from detergents, proteins, salts and
reagents used during cell lysis step.
Precipitate DNA with an alcohol usually 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.

Separation of DNA From Proteins and Lipids

Phenol extraction:
Add 1 vol phenol to 1 vol of aqueous solution; mix to get an emulsion
Add 1/2 vol (relative to aqueous solution) of chloroform
(to improve phase separation) and mix
Spin

Phase separation
- proteins partition to phenol (Organic Phase) and interphase,
form a white layer in interphase
- while DNA is in aqueous phase (normally the upper layer)
Repeat if necessary

Separation of DNA From Proteins and Lipids

Phenol extraction (cont)

Add to aqueous phase 1 vol of chlorofom, mix

and spin (to remove phenol traces)
EtOH precipitate DNA to concentrate and
remove traces of phenol/chlorofom

Critical Parameters
Need to minimize activity of endogenous nucleases
Freeze tissue
EDTA in solubilization buffer

Minimize shearing of DNA

Gentle (but thorough) mixing
Avoid EtOH pptn and instead remove organic
solvents and salt from DNA by dialysis

Disruption of starting material

(Virus, bacteria, plant or animal cell)
Lysis Buffer
What is Lysis Buffer?
50 mM Tris-HCI, pH 8.0
1% SDS
Tris buffer to maintain the pH of the solution at a
level where DNA is stable

1% SDS to break open the cell and nuclear

membranes, allowing the DNA to be released into
the solution (SDS also denatures and unfolds
proteins, making them more susceptible to
protease cleavage).

Deproteinization
Why Add Protease?
Protease is added to destroy nuclear proteins
that bind DNA and cytoplasmic enzymes that
breakdown and destroy DNA.
Protease treatment increases the amount of
intact DNA that is extracted.

Adding Salt

The protease solution already contains salt

Na+ ions of NaCI bind to the phosphate groups of DNA
molecules, neutralizing the electric charge of the DNA molecules.
The addition of NaCI allows the DNA molecules to come
together instead of repelling each other, thus making it easier
for DNA to precipitate out of solution when alcohol is added.

Adding Ice Cold Alcohol?

DNA does not dissolve in alcohol.

The addition of cold alcohol makes the DNA clump

together and precipitate out of solution.

Precipitated DNA molecules appear as long pieces of

fluffy, stringy, web-like strands.

Microscopic oxygen bubbles aggregate , or fuse

together, as the DNA precipitates.

The larger, visible air bubbles lift the DNA out of

solution, from the aqueous into the organic phase.

DNA,RNA
Solution
Denatured
Protein
Phenol
Cell Extract

Shake

Separate layers
by centrifugation

Cells
Extract

Bacterial Cells
Or tissue culture cells
Or blood
Or flies..

HOW?
Pure DNA

TYPES OF METHODS:
Differential solubility
Adsorption methods

Density gradient centrifugation

General Features:
Denaturing cell lysis (SDS, alkali, heating)
Enzyme treatments

- protease
- RNase (DNase-free)
- DNase (RNase-free)

Purifying one type of DNA

away from other DNA molecules

-Plasmids from bacterial chromosomal DNA

-phage DNA

1. Differential solubilization

SDS, alkali

Differential
solubility

alkali

neutralize

2.Density Gradient
Centrifugation

Plasmid DNA

3. Adsorption Method

Resins (glass or chemically modified beads) that bind

nucleic acids reversibly are packaged in columns for
easy DNA & RNA purification

Anion exchange
chromatography

Preparation of Genomic DNA From

Mammalian Tissue
Freeze tissue Liquid nitrogen or dry ice
Crush to produce digestible pieces
Solubilise with buffer containing SDS and proteinase K
(to digest most of cellular proteins)
Separate from proteins by successive phenol/chlorofom
extractions
Recover DNA by dialysis/EtOH pptn

Quality of Genomic DNA

Size: bigger than 100Kb
At 260nm 1 O.D. = 50g/ml dsDNA
Purity : index A260/A280
Ratio larger than 1.8 high quality DNA
Ratio of 1.5 indicates soln of 50% DNA 50% protein

Quantifying the DNA

The amount of DNA can be quantified using the formula:
DNA conc. (g/ml)= OD260 x 100 (dilution factor) x 50 g/ml
1000
Nucleic acids have a peak absorbance in the ultraviolet range
at about 260 nm

1 A260 O.D. unit for dsDNA = 50 g/ml

1 A260 O.D. unit for ssDNA = 33 g/ml

1 A260 O.D. unit for RNA = 40 g/ml

HIGH MW GENOMIC DNA ISOLATION

TYPICAL PROCEDURE
1 Cell Lysis
0.5% SDS + proteinase K (55oC several hours)
2 Phenol Extraction
gentle rocking , few times
3 Ethanol Precipitation
4 RNAse followed by proteinase K
5 Repeat phenol extraction and EtOH ppt

Laboratory Protocol
Mini-prep Protocol
1.

Grow bacterial colonies overnight in LB with antibiotic (1-5 ml cultures)

2.

Take out 1.5 ml bacteria and spin down for 2 min. Remove supernatant.

3.

Add 100 ul Solution 1. Vortex to resuspend pellet.

4.

Add 200 ul Solution 2. Vortex and wait 1-2 min.

5.

Add 150 ul Solution 3. Vortex.

6.

Spin 10 min at 12,000 rpm at room temperature.

7.

Remove the supernatant and put in new tube.

8.

Add 3X volume of 95% Ethanol (Preferably cold Ethanol).

9.

Invert to mix and spin for 10 min at room temperature.

There should be a white pellet now.

10. Discard supernatant.

11. Add 500 ul 70% Ethanol. Spin 2 min, and remove supernatant
12. Dry pellet, (in the hood or in open environment).

13. Resuspend in 20-30 ul TE.

Making Solution
Solution 1
50 mM glucose
25 mM Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)

0.9 g glucose
2.5mL 1M Tris-Cl (pH 8.0)
2.0 ml 0.5M EDTA (pH 8.0)
Water up to 100 ml

Solution 2
0.2 N NaOH
1% SDS

2 ml 1N NaOH
1 ml 10% SDS
Water up to 10 ml

Solution 3
5 M potassium acetate
11.5% acetic acid

60 ml 5M KoAc
11.5 ml glacial acetic acid
Water up to 100 ml

Places where students (but certainly not you)

will mess this up
1.

Losing track of what you have or have not

added (see organization chart)

2.

Not labeling tubes properly.

3.

Waiting too long to add Proteinase K

4.

Not changing pipette tips

5.

Throwing out their eluted DNA (yes, this is a

common mistake!)

RNA Extraction
Principles of DNA & RNA purification
Are similar but RNA is easily degraded
Chemically and enzymatically

Isolation of mRNA
Required for gene cloning and expression analysis
Major difficulty in RNA isolation is that most ribonucleases
(RNases) are very stable and active enzymes that require no
cofactors to function
Therefore, first step in RNA isolation is to lyse cells in the
presence of chemicals that will denature ribonucleases
Crucial that denaturant in contact with cellular contents at
moment of disruption as RNA unstable as harvest begins

Isolation of mRNA
All solutions DEPC (diethylpyrocarbonate) treated

Lyse in the presence of RNase inhibitors

e.g. placental ribonuclease inhibitor (RNAsin)
Lyse cells and release cytosol, pellet nuclei/membranes,
then phenol extract and EtOH pptn
If have contaminating DNA can remove with
RNase-free DNase I
RNA suspended & stored in safe RNase-free soln

RNA PURIFICATION
Lyse & denature proteins FAST

USES OF ISOLATED DNA/RNA

Preparation of genomic libraries/cDNA libraries

PCR template

Cloning

Gene/DNA sequencing

Analysis of genomic organization

Study gene structure

DNA fingerprinting

Analysis of genome composition

Detection of abnormalities / mutations