DNA and RNA extraction

1. DNA extraction

2. Basics of Molecular biology

3. Molecular biology: definition
• Molecular biology is the study of the process of replication,
transcription and translation of the genetic material.

4. Components involve in molecular
biology
DNA
RNA
Protein

5. Deoxyribonucleic acid (DNA)
• DNA is a nucleic acid that contains the genetic
instructions used in the development and functioning of
all known living organisms and some viruses.

6. • Two long strands makes
the shape of a double helix.
• two strands run in opposite
directions to each other and
are therefore anti-parallel.
• Chemically, DNA consists
of two long polymers of
simple units called
nucleotides, with
backbones made of base,
sugars and phosphate
groups.
Fig : DNA double helix

7. nucleoside
Sugar +Base = nucleoside
Phosphate+ sugar + Base = nucleotide

8. • Types:- adenine and guanine (fused five- and six-
membered heterocyclic compounds) – Purines
• cytosine & thymine (six-membered rings)-Pyrimidines.
• A fifth pyrimidine base, called uracil (U), usually takes
the place of thymine in RNA and differs from thymine by
lacking a methyl group on its ring.
• PAIRING : A =T and A=U
G≡C
Bases

9. Ribonucleic acid (RNA)
• RNA is a biologically important type of molecule that consists of a
long chain of nucleotide units.
• Each nucleotide consists of a nitrogenous base, a ribose sugar, and
a phosphate.

10. Types of RNA
Type Abbr Function Distribution
Messenger RNA
mRNA Codes for protein All organisms
Ribosomal RNA rRNA Translation All organisms
Transfer RNA tRNA Translation All organisms

13. Basic players in molecular biology: DNA, RNA, and
proteins. What they do is this :

14. DNA isolation
DNA must be separated from proteins and cellular debris.
Separation Methods
 Organic extraction
 Salting out
 Selective DNA binding to a solid support

15. Organic extraction
 DNA is polar and therefore insoluble in organic solvents.
 Traditionally, phenol:chloroform is used to extract DNA.
 When phenol is mixed with the cell lysate, two phases
form. DNA partitions to the (upper) aqueous phase,
denatured proteins partition to the (lower) organic phase.
 DNA is a polar molecule because of the negatively
charged phosphate backbone.
 This polarity makes it more soluble in the polar aqueous
phase.

16. Genomic DNA isolation: phenol extraction
1:1 phenol : chloroform
 Phenol: denatures proteins, precipitates form at interface
between aqueous and organic layer
 Chloroform: increases density of organic layer
 Isoamyl alcohol: prevents foaming

17. Genomic DNA isolation: phenol extraction

18. Binding to a support material
Most modern DNA purification methods are based on
purification of DNA from crude cell lysates by selective
binding to a support material.
Support Materials
 Silica
 Anion-exchange resin
Advantages
 Speed and convenience
 No organic solvents
 Amenable to automation
Disadvantage
 DNA fragmentation

19. Silica matrix based genomic DNA
isolation

20. Concentration measurement
Photometric measurement of DNA concentration
UV 260 nm
Conc=50xOD260

21.  DNA extraction is needed for genetic analysis which
used for:
 1- scientific: use DNA in number of Applications , such as
introduction of DNA into cells & animals or plants for
diagnostic purposes (gene clonining)
 2- Medicine: is the most common. To identify point sources for
hospital and community-based outbreaks and to predict
virulence of microorganisms
 3- forensic science: needs to recover DNA for identification of
individuals ,( for example rapists, petty thieves, accident , or
war victims) , and paternity determination.

22.  Many different methods and technologies are
available for the isolation of genomic DNA.
 All methods involve:
 A. disruption and lyses of the starting material
followed by
 B. Removal of proteins and other contaminants and
finally
 C. Recovery of the DNA

23. Why purifying genomic DNA?
 Many applications require purified DNA.
 Purity and amount of DNA required (and process used)
depends on intended application.
 Example applications:
 Tissue typing for organ transplant
 Detection of pathogens
 Human identity testing
 Genetic research

24. DNA purification challenges
1. Separating DNA from other cellular components such
as proteins, lipids, RNA, etc.
2. Avoiding fragmentation of the long DNA molecules by
mechanical shearing or the action of endogenous
nucleases.
Effectively inactivating endogenous nucleases (DNase
enzymes) and preventing them from digesting the
genomic DNA is a key early step in the purification
process. DNases can usually be inactivated by use of
heat or chelating agents.

25. DNA purification
There are many DNA purification methods. All must:
1. Effectively disrupt cells or tissues
(usually using detergent)
2. Denature proteins and nucleoprotein complexes
(a protease/denaturant)
3. Inactivate endogenous nucleases
(chelating agents)
4. Purify nucleic acid target away from other nucleic acids
and protein
(could involve RNases, proteases, selective matrix and alcohol
precipitations)

26. Sample Collection
Sample can be isolated from any living or dead
organism
Common sources for DNA isolation include:
 Whole blood
 Buffy coat
 Bone material
 Buccal cells
 Cultured cells
 Amniocytes or amniotic fluid
 Sputum, urine, CSF, or other body fluids

27.  Key Steps
 Lysis of the cells
 Removal of contaminants
includes
 Proteins
 RNA
 Other macromolecules
 Concentration of purified
DNA

28. Magnetic beads with DNA binding capacity
 Magnetic beads are coated with DNA antibodies
or silica to bind to DNA.
 Samples are lyses & and then treated with
proteinase K.
 The lysates are then applied to the beads.
 Resin is subsequently washed & DNA is eluted of it
at 65c
 Magnetic beads are separated from the sample
on a magnetic stand.

29. Use Detergent to solubilize the membrane lipid.

30. 2. Separate DNA From Crude
Lysate
 DNA must be separated from proteins and cellular
debris.
Separation Methods
a) Organic extraction
b) Salting out
c) Ethanol precipitation:
-Precipitation of DNA: Absolute Ethanol is layered on the top
of concentrated solution of DNA

31. Solid Phase Isolation
 The diagram below explains the attractive
properties of solid phase for DNA and RNA.

32. DNA Purification Evaluation
 DNA concentration can be determined by measuring
the intensity of absorbance with a
spectrophotometers & comparing to a standard
curve of known DNA concentration.
 Measuring the intensity of absorbance of the DNA
solution at wavelength 260nm & 280nm is used as a
measure of DNA purity
 DNA purity: A260/A280 ratio: 1.7 – 1.9
 DNA concentration (μg/ml): A260 X 50
 DNA yield:
DNA conc. X Total volume of DNA solution

33. Spectrophotometers