Gene & Cell Technology ABS-831

Course Introduction

What is a cell?

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

Prokaryotic cell

Eukaryotic cell

Structure of a 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

Plasma membrane and membranes of

organelles (nuclear envelope included)

DNA located in nucleus

A lot of proteins around

Mitochondrial DNA

Structure of the cell

Goals:

Removal of proteins

DNA vs RNA

Isolate specific type of nucleic acid

Isolation of Nucleic Acids

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

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

HOW?

Extract

Cells

Pure DNA

General Features:

Denaturing cell lysis (SDS, alkali, heating)

Enzyme treatments

- protease

- RNase (DNase-free)

- DNase (RNase-free)

TYPES OF METHODS:

Differential solubility

Adsorption methods

Density gradient centrifugation

Purifying one type of DNA away from other DNA molecules

-Plasmids from bacterial chromosomal DNA

-phage DNA

SDS, alkali

1. Differential solubilization

alkali

neutralize

Differential

solubility

Plasmid DNA

2.Density Gradient Centrifugation

Resins (glass or chemically modified beads) that bind

nucleic acids reversibly are packaged in columns for

easy DNA & RNA purification

3. Adsorption Method

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

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

HIGH MW GENOMIC DNA ISOLATION

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.

Solution 1 50 mM glucose 0.9 g glucose 25 mM Tris-Cl (pH 8.0) 2.5mL 1M Tris-Cl (pH 8.0) 10 mM EDTA (pH 8.0) 2.0 ml 0.5M EDTA (pH 8.0) Water up to 100 ml

Solution 2 0.2 N NaOH 2 ml 1N NaOH 1% SDS 1 ml 10% SDS Water up to 10 ml

Solution 3 5 M potassium acetate 60 ml 5M KoAc 11.5% acetic acid 11.5 ml glacial acetic acid Water up to 100 ml

Making Solution

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!)

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

RNA Extraction

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