Gene Manipulation and Genetic

Engineering
Principles of Nucleic Acid Isolation

Instructor: Dr. Mohsin Gulzar Barq

Assistant Professor
Course Code: LT3302
 CONTENTS
• Introduction and Scope
• Principles of Nucleic Acid Isolation
• Restriction and Modification System
• Properties of restriction endonucleases, their occurrence and
recognition sequences. Assay procedures for restriction
endonucleases and slab gel electrophoresis. Practical uses of
endonucleases. Role in genetic engineering
• Cloning vehicles: plasmids, cosmids and phagemids, YAC and
BAC etc. Construction of genomic and cDNA libraries.
Cloning strategies. Methods of introducing exogenous DNA.
Methods for screening the clones.
• DNA sequencing. PCR its application. Introduction to
expression systems for Prokaryotes and Eukaryotes.
• Recombinant Protein Production. How to make transgenic and
gene knockouts
 THE CENTRAL DOGMA OF
MOLECULAR BIOLOGY

non-coding RNA (rRNA, tRNA, siRNA, etc.)

Transcription
Replication

mRNA

Translation
Deoxyribonucleotide

Ribonucleotide
 APPLICATIONS
DNA

Amplification methods (PCR, LCR)

Restriction enzyme digest
Hybridization methods (Southern analysis)
Sequencing
RNA

Amplification methods (RT-PCR)

Hybridization methods (Northern analysis)
The very first DNA isolation was done by a Swiss physician,
Friedrich Miescher in 1869
FUNDAMENTAL STEPS OF DNA
PURIFICATION
 Sample Lysis
 Removal contaminants from Nucleic acids
 Concentration of Nucleic acids
 Measurement of purity and concentration of Nucleic acids
 DNA PURIFICATION: OVERVIEW

Cell growth Cell harvest and lysis

DNA concentration DNA purification

 CELL LYSIS
Mechanical Method
Grinding in Liquid N2
Sonication
Homogenization
Heat

Chemical method
SDS (Sodium Dodecyl Sulphate
Triton X-100
CTAB (Cetyl trimethyl ammonium bromide also called
Cetrimonium bromide)

Enzymatic Method
Lysozyme
Zymolase & Murienase
Proteinsae K
SEPARATION OF NUCLEIC ACIDS
FROM THE CONTAMINANTS
Organic (Phenol-Chloroform) Extraction
Non-Organic (Proteinase K and Salting out)
Chelex (Ion Exchange Resin) Extraction
Silica Based
FTA Paper (Collection, Storage, and Isolation) (Quiz or
Assignment)
ORGANIC SOLVENT EXTRACTION
METHOD
 Adsorption Chromatography Method
Step 1: Prepare crude lysate Step 2: Adsorb to silica surface

Apply to column Centrifuge

Nucleic acids
Silica-gel membrane

Extraction Buffer composition favors Flow through

DNA and RNA adsorption to silica: (discard)
• Low pH
• High ionic strength
• Chaotropic salt Nucleic acids bind to the membrane,
while contaminants pass through the
column.

Surface silanol groups are weakly

acidic, and will repel nucleic acids at
near neutral or high pH due to their
negative charge
Adsorption Chromatography Method

Step 3: Wash away residual contaminants

Centrifuge
Wash buffer

Nucleic acids Nucleic acids

Flow through
(discard)
Step 4: Elute nucleic acids

Centrifuge
Elution buffer

Nucleic acids

Elution Buffer composition is unfavorable to

surface binding:
High pH
Low ionic strength
Nucleic acids
 FTATM Paper Extraction

• Cellulose based storage paper

• Extraction/storage of nucleid acids from

blood, buccal cells, tissue, cultured cells,
microorganisms, plant tissue and other..

• DNA is stable at room temperature for years

Fast and efficient DNA preparation:
punch and purify
• Apply sample on FTA paper and let dry

• Cells are lysed on contact

• Use punch to add sample to tube

• Wash to remove PCR inhibitors

• Add “punch“ directly to PCR reaction

CONCENTRATION OF THE gDNA
Ethanol depletes the hydration shell surrounding
DNA and Reduces repulsive forces between DNA
strands which causes aggregation and precipitation
of DNA

70% final conc.

“spooling” Ethanol precipitation

PLASMIDS: VEHICLES OF
RECOMBINANT DNA
Bacterial cell

Genomic DNA Plasmids

Non-chromosomal DNA
Replication: independent of the chromosome
Many copies per cell
Easy to isolate
Easy to manipulate
Plasmid Purification: Alkaline lysis

Alkaline conditions
denature DNA

Neutralize: genomic
DNA can’t renature
(plasmids can because
they never fully
separate)
ISOLATION OF RNA
• Requires Strict precautions to avoid sample
degradation
• RNAses are enzymes which are small proteins that
can renature and become active
• Must be eliminated or inactivated before isolation
• Critical to have separate RNAse free lab area
PROBLEMS WITH RNA
RNA is chemically unstable -- spontaneous cleavage of
phosphodiester backbone via intramolecular transesterification
( group of an alcohol is exchanged with an R' group of an ester)

RNA is susceptible to nearly ubiquitous RNA-degrading

enzymes (RNAses)

RNAses are released upon cell lysis

RNAses are present on the skin
RNAses are very difficult to inactivate
-- disulfide bridges conferring stability
Common sources of RNase and
how to avoid them
Contaminated solutions/buffers

 Use good sterile technique

 Treat solutions with Diethyl pyrocarbonate (DEPC)
 Make small batches of solutions

Contaminated equipment

 Use “RNA-only” pipettes, glassware, gel rigs

 Bake glassware, 300°C, 4 hours
 Use “RNAse-free” pipette tips
 Treat equipment with DEPC
Top 10 sources of RNAse contamination
(Ambion Scientific website)

1) Ungloved hands
2) Tips and tubes
3) Water and buffers
4) Lab surfaces
5) Endogenous cellular RNAses
6) RNA samples
7) Plasmid preps
8) RNA storage (slow action of small amounts of RNAse
9) Chemical nucleases (Mg++, Ca++ at 80°C)
10) Enzyme preparations
INHIBITORS OF RNAse
DEPC

 Diethylpyrocarbonate
 Alkylating agent, modifying proteins and nucleic acids
 Fill glassware with 0.1% DEPC, let stand overnight at room
temp.
 Solutions may be treated with depc -- add depc to 0.1%, then
autoclave (DEPC breaks down to CO2 and ethanol)
Making and using mRNA (1)
Making and using mRNA (1)
Terms

• Western Blotting

• Northern Blotting

• Southern Blotting

• Eastern Blotting: Different types of probes to detect post-

translational modifications of proteins

• S1 nuclease mapping is a nuclease protection assay using

nuclease S1. This technique is used to quantify and map RNA
transcripts. It is capable of identifying individual RNAs in a
mixture of RNA sample of known sequence
PURIFYING RNA: THE KEY TO SPEED

Break the cells/solubilize components/inactivate RNAses

by the addition of guanidinium thiocyanate (very powerful
denaturant)
Extract RNA using phenol/chloroform (at low pH)
Precipitate the RNA using ethanol/LiCl
Store RNA:
in DEPC-treated H20 (-80°C)
in Formamide (deionized) at -20°C
Selective capture of mRNA:
Oligo dT-cellulose

Oligo dT is linked to cellulose matrix

RNA is washed through matrix at high salt concentration

Non-polyadenylated RNAs are washed through

polyA RNA is removed under low-salt conditions

• (not all of the non-polyadenylated RNA gets removed)

Other methods to capture mRNA
 Poly(U) sepharose chromatography

 Poly(U)-coated paper filters

 Streptavidin beads:
• A biotinylated oligo dT is added to guanidinium-treated cells, and
it anneals to the polyA tail of mRNAs
• Biotin/streptavidin interactions permit isolation of the
mRNA/oligo dT complexes
• Streptavidin is a tetrameric biotin-binding protein that is isolated
from Streptomyces avidinii and has a mass of 60,000 daltons
• Water soluble B Vitamin
NUCLEIC ACID ANALYSIS

• DNA or RNA is characterized using several different methods for

assessing quantity, quality, and molecular size.

• UV spectrophotometry
• Agarose gel electrophoresis
• Fluorometry
• Colorimetric blotting
 QUANTIFICATION FROM UV
SPECTROPHOTOMETRY

• DNA and RNA absorb maximally at 260 nm.

• Proteins absorb at 280 nm

• [dsDNA] = (A260) X dilution factor X 50 µg/mL

• [ssDNA] = (A260) X dilution factor X 33 µg/mL
• [RNA] = (A260) X dilution factor X 40 µg/mL
• [Oligonucleotides] = (A260) X dilution factor X 20-30µg/mL
 QUALITY FROM UV
SPECTROPHOTOMETRY
 The A260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for
ssRNA
 Ratios lower than 1.7 usually indicate significant protein
contamination.
 The A260/A230 ratio of DNA and RNA should be roughly
equal to its A260/A280 ratio (and therefore ≥ 1.8)
 Lower ratios may indicate contamination by organic
compounds (e.g. phenol, alcohol, or carbohydrates)
 QUALITY FROM AGAROSE GEL
ELECTROPHORESIS

• Genomic DNA:
• 0.6% to 1% gel, 0.125 µg/mL Ethidium bromide in gel
and/or in running buffer
• Electrophorese at 70–80 volts, 45–90 minutes

• Total RNA:
• 1% to 2% gel, 0.125 µg/ml Ethidium bromide in gel and/or
in running buffer
• Electrophorese at 80–100 volts, 20–40 minutes
DNA Size from Agarose Gel Electrophoresis:
Compares unknown DNA to known size
standards
Lambda DNA cut 1 kb ladder
Lambda DNA with Hind III
12,218 bp 100 bp ladder
6,018 bp
23,130 bp
1,500 bp
9,416 bp 3,054 bp 1,000 bp
6,557 bp 2,036 bp 600 bp

4,361 bp 1,636 bp
300 bp
48,500 bp 1,018 bp
(48.5 kb)
100 bp
2,322 bp
2,027 bp 517 bp
 DNA Quality from Agarose Gel
Electrophoresis
Human Whole Blood DNA

Lambda DNA
marker Whole blood genomic DNA
Lambda DNA cut with
Hind III marker
 CULTURED CELL RNA

DNA

28S
18S

100 50 25 ng
5S rRNA, tRNA,
Genomic DNA and other small
Degraded RNA
markers RNA molecules
mRNA = background smear
high  low MW
FLUOROMETRY

• Fluorometric DNA quantitation instruments and assays are

highly selective for dsDNA (double-stranded DNA)
• Fluorometric methods use intercalating fluorescent dyes (SYBR
Green) that bind specifically to dsDNA molecules
• Fluorometric instruments detect fluorescent signals from bound
dyes and estimate dsDNA concentration relative to calibration
reagents
CALORIMETRIC BLOTTING

• The concentration of a DNA sample can be determined using

diphenylamine method using a colorimeter or a
spectrophotometer
• In this method a set of standards (where the concentration of
DNA is known) is used and the concentration of the unknown
sample is then determined by comparing it with the standard
 STORAGE CONDITIONS

• Store DNA in TE buffer at 4 °C for weeks or at –20 °C

to –80 °C for long term.
• Store RNA in RNase-free ultra pure water at –70 °C.