The document discusses the isolation and purification of genomic DNA and plasmid DNA. It begins by outlining the history of DNA isolation and describes current methods. The key steps in isolating genomic DNA are cell growth and lysis, followed by DNA purification using techniques like phenol-chloroform extraction and ethanol precipitation to remove proteins and RNA. Plasmid DNA isolation involves bacterial growth, lysis, and purification of plasmids from chromosomal DNA using alkaline lysis and centrifugation. Purification methods like silica binding columns are also described to further clean DNA samples.

1. UNIVERSITY OF ALLAHABAD
DEPARTMENT OF BOYANY
ISOLATION & PURIFICATION OF GENOMIC DNA AND
PLASMID DNA
Under the Supervision
Prof . S.M. Prasad
Presented by
Aditya Kumar
M.Sc. III Sem. Botany

2. 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 .

3. 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.

4. 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 .

5. 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 cheek
Plant 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 .

7. 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.

8. 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

9. cell growth
cell harvest
and lysis
DNA purification
DNA purification: overview
DNA concentration

10. 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.

11. • Tow general types of procedure are used for the purification of DNA
Centrifugation
Chemical extraction
The principal of centrifugation is as follows
The sample is spun at high speed and centrifugation force causes the
large 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 .

13. • 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 :-

14. DNA purification: phenol/chloroform extraction
1:1 phenol : chloroform
or
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

15. 1. Aqueous volume (at least 200 microliters)
2. Add 2 volumes of phenol:chloroform, mix well
3. Spin in centrifuge, move aqueous phase to a new tube
4. Repeat steps 2 and 3 until there is no precipitate at
phase interface
5. (extract aqueous layer with 2 volumes of chloroform)
Phenol extraction

16. 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)

17. DNA purification: silica binding
Binding occurs in presence of high
salt concentration, and is disrupted
by elution with water

19. 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

20. 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 minute
6-wash column and spin twice
7- elute DNA

21. 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..

24. 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.

25. Isolation and Purification of plasmid DNA
Extraction 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

26.  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.

27. 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.

28. 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.

29. 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.

30. Plasmid purification: alkaline lysis
Alkaline
conditions
denature
DNA
Neutralize:
genomic DNA
can’t renature
(plasmids
CAN because
they never
fully
separate)

31. 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.

32. • 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.

33. 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.

34. 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

36. 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

37. 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).

39. Video protocall

40. 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.