UNIVERSITY OF ALLAHABAD DEPARTMENT OF BOYANY

ISOLATION & PURIFICATION OF GENOMIC DNA AND PLASMID DNA

Under the SupervisionProf . S.M.

Prasad

Presented by Aditya Kumar

M.Sc. III Sem. Botany

Introduction :-DNA was first isolated in 1869 by a post doctoral

student Friedrich Miescher at the University of Tubingen.

• Friedrich Miescher obtained his first DNA, which he referred to it as nuclein, from human leukocytes washed from pus-laden .

Cont….Molecular manipulation of specific genes is one of

the very popular areas of research in biotechnology .These genes therefore ,should be either or artificially

synthesized before they are manipulated and used for transformation leading to the production of transgenic animals and in plant.

Therefore we discuss the gene technology available for the isolation and the purification of genomic DNA and plasmid DNA.

Genomic DNA Genome :- The sum of the total of all genetic material of an organism which store biological information are called Genome .The nature of the genome may be either DNA or RNA .All eukaryotes and prokaryotes always have a DNA genome

these are called the type of genomic DNA but viruses may either have a DNA genome and RNA genome .

Isolation of genomic DNA DNA was first isolated in 1869 by a post doctoral student Friedrich

Miescher .DNA isolation was a long and difficult process these are isolate from

different part of animals plants and bacteria and viruses Nowadays for examples enough DNA can be collected for genetic

manipulations in laboratory mice or rats from a small pieces of the tail .Human DNA analysed using small blood samples or a few cells scraped

from the inside of the cheekPlant cells have very rigid cell wall the scientist must mechanically

break the cells open the blender or add special degradative enzyme to digest the cell component .

Cont…The method of isolation of genomic DNA from comprises following steps. 1. Bacterial culture growth and harvest. 2. Cell wall rupture and cell extract preparation 3. DNA Purification from the cell extract. 4. Concentration of DNA solution.

Purification of DNA

Definition :-• DNA purification is a

technique that removes impurities and unused reagents from samples by using enzymatic reaction and other instruments such as PCR and electrophoresis etc.

Before (b) and after (a) DNA purification

DNA

Impurities

www1.qiagen.com

cell growth cell harvest and lysis

DNA purification

DNA purification: overview

DNA concentration

ISOLATION AND PURIFICATION OF DNA

DNA can be isolated by removing the cell wall and cell membrane components then proteins are removed by phenol and finally the RNA is removed by ribonuclease.

• Tow general types of procedure are used for the purification of DNA Centrifugation Chemical extraction The principal of centrifugation is as followsThe sample is spun at high speed and centrifugation force causes thelarge or heavier components to sediments in to the bottom of the tube When the sample is centrifuged DNA and some other large

components are sediments to the bottom of the tube .The fragments of cell wall together with many other soluble

components remain in solution and are discarded .

• The sedimented DNA is then resolved in an appropriate buffer solution However ,it still has a lot of protein and RNA mixed in with it.

These are generally removed chemical means :-

DNA purification: phenol/chloroform extraction

1:1 phenol : chloroformor

25:24:1 phenol : chloroform : isoamyl alcohol

Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer

Chloroform: increases density of organic layer

Isoamyl alcohol: prevents foaming

1. Aqueous volume (at least 200 microliters)2. Add 2 volumes of phenol:chloroform, mix well3. Spin in centrifuge, move aqueous phase to a new

tube4. Repeat steps 2 and 3 until there is no precipitate

at phase interface5. (extract aqueous layer with 2 volumes of

chloroform)

Phenol extraction

Ethanol depletes the hydration shell surrounding DNA…• Allowing cations to interact with the DNA phosphates• Reducing repulsive forces between DNA strands• Causing aggregation and precipitation of DNA

• Aqueous volume (example: 200 microliters)-- add 22 microliters sodium acetate 3M pH 5.2-- add 1 microliter of glycogen (gives a visible pellet)-- add 2 volumes (446 microliters) 100% ethanol-- mix well, centrifuge at high speed, decant liquid-- wash pellet (70% ethanol), dry pellet, dissolve in

appropriate volume (then determine DNA concentration)

Ethanol precipitation (DNA concentration)

DNA purification: silica binding

Binding occurs in presence of high salt concentration, and is disrupted by elution with water

Purification of mammalian genomic DNA

1-Release DNA Enzymatic digestion 2-Prepair column Add solution and spin 3-Bind DNA Add ethanol and spin 4-Wash DNA Wash and spin 5- Spin DNA 1 minute

Purification of plant genomic DNA:- 1-Release DNA

Enzymatic digestion 2-Prepair column Add solution and spin 3-Bind DNA Add ethanol and spin 4-Wash DNA Wash and spin 5- Spin DNA 1 minute6-wash column and spin twice7- elute DNA

DNA Purification by column method :- In this purifying DNA by passing it through a

column containing a resin that binds DNA but not other the components The two main choices are silica and anion exchange resins .Silica resins bind nucleic acids rapidly and specifically at low pH and high salt concentration .The nucleic acids are released at higher pH low salt concentration.Anion exchange resins are positively charged and bind DNA but its

negatively charged phosphate groups .In this case binding occurs at low salt concentration and the nucleic acids

are eluted by conc. Of salt ,which disrupt the ionic bonding..

Plasmid DNA Plasmids are defined as autonomous elements whose genomes exist in the cell as extra chromosomal units. They are self replicating circular (rarely linear) duplex DNA molecules ,which are maintained in a characteristics number of copies in a bacterial cell , yeasts cell or even in organelles found in eukaryotic cells.

Isolation and Purification of plasmid DNAExtraction of plasmid DNA bacteria are require that the cellular

contents be liberated into a solution For bacteria, an enzyme called lysozyme digest the peptidoglycan ,

which is the main component of the cell wall.A successive treatment with detergents dissolves the lipids of the cell

wall membrane.Chelating agents such as EDTA (Ethylene diamine tetraacetate) are

also used ,especially gram –ve bacteria

Proteins are removed, the sample still contains RNA along with the DNA because it is also a nucleic acid, it is not soluble in phenol.

The enzyme ribonuclease (Rnase) digest RNA into ribonucleotides. Ribonuclease treatment leaves a sample of DNA in a solution containing short pieces of RNA and ribonucleotide.

The isolation of plasmid DNA involves three major steps- 1. Growth of the bacterial cell. 2. Harvesting and lysis of the bacteria. 3. Purification of the plasmid DNA.

Growth of the bacterial cell It involves growth of the bacterial cells in a media containing essential

nutrients.

Harvest and lysis of bacteria Lysis of bacteria results in the precipitation of DNA and cellular

proteins.Addition of acetate-containing neutralization buffer results in the

precipitation of large and less supercoiled chromosomal DNA and proteins leaving the small bacterial DNA plasmids in solution.

Plasmid DNA Purification

Mix sample and buffer. Makes DNA in solution ready to bind column.

Load onto column. Nucleic acids bind to membrane. Other components flow through.

Wash Column. Removes any contaminants.

Elute DNA. DNA releases from column and is ready for downstream applications.

Plasmid purification: alkaline lysis

Alkaline conditions denature DNA

Neutralize: genomic DNA can’t renature (plasmids CAN because they never fully separate)

Purification of Plasmid DNA

• A rapid method for making a small preparation of purified plasmid DNA from culture volumes as low as 1 mL is called a miniprep.

• Transformed cells from an antibiotic-resistant colony are grown to stationary phase in an overnight suspension culture.

• The cells are collected by centrifugation and resuspended in a buffered solution of glucose and ethylene diamin etetraacetic acid (EDTA), which binds divalent cations (such as Mg++ and Ca++) necessary for cell membrane stability.

• The suspended cells are then treated with a mixture of SDS and sodium hydroxide.

SDS, an ionic detergent, dissolves the phospholipid and protein components of the cellular membrane.

This lyses the membrane, releasing the cell contents. Sodium hydroxide denatures both plasmid and

chromosomal DNAs into single strands. The chromosomal DNA separates completely into

individual strands; however, the single-stranded plasmid loops remain linked together like interlocked rings.

Subsequent treatment with potassium acetate and acetic acid forms an insoluble precipitate of SDS/lipid/protein and neutralizes the sodium hydroxide from the previous step.

At neutral pH, DNA renatures. In the miniprep, the long strands of chromosomal DNA only partially renature and become trapped in the SDS/lipid/protein precipitate.

The linked, single-stranded plasmid DNA completely renatures into double-stranded molecules that remain in solution and largely escape entrapment in the precipitate.

The precipitate is pelleted by centrifugation and discarded, leaving the plasmid DNA (as well as RNA molecules) in the supernatant.

Ethanol or isopropanol is added to the supernatant to precipitate the plasmid DNA out of solution.

The plasmid DNA is pelleted by centrifugation, washed with ethanol, dried, and resuspended in a small volume of buffer.

Subsequent treatment with RNase destroys RNA, leaving relatively clean plasmid DNA

Plasmid Isolation from Bacteria

(a) Inoculation and harvesting the bacteria

(b) lysis of the bacteria (heat, detergents (SDS or Triton-114), alkaline(NaOH)),(c) neutralization of cell lysate and separation of cell debris (by centrifugation),

Or other cell types

Cont..Traditional Ways

(d) collecting plasmid DNA by centrifugation (after ethanol precipitation or through filters - positively charged silicon beads),

(e) check plasmid DNA yield and quality (using spectrophotometer and gel electrophoresis).

Video protocall

Advantage Most of the RNA and protein under defined conditions (specifically

cell lysis by SDS and neutralization with sodium acetate) can be removed by the centrifugation.

THANK

YOU……