Molecular Genetic

Importance of Molecular Genetics

 Genetics is playing an important role in the practice of

clinical medicine.
 - Medical genetics involves any application of genetics
to medical practice, it thus includes:
 Studies of the inheritance of disease in families.
 Mapping of disease genes to specific locations on
chromosomes
 Analysis of the molecular mechanisms through which
genes cause disease
 Diagnosis and treatment of genetic disease (ex. Gene
therapy)
 DNA Isolation
• DNA isolation: is an extraction process of
DNA from various sources.

• The aim: is to separate DNA present in the

nucleus of the cell from other cellular
components.
 Application of DNA isolation
 It 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.
 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
• To choice of a method depends on many
factors:

A. The quantity and molecular weight of the

DNA
B. The purity required for application
C. The time and expense
 Sample Collection
A- Source: 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
DNA Purification & Quantification
• Separating DNA from other cellular components
such as proteins, lipids, RNA, etc.
• 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.
• Key Steps
• Lysis of the cells
• Removal of contaminants
includes
 Proteins
 RNA
 Other macromolecules
• Concentration of purified
DNA
Use Detergent to solubilize the membrane lipid.
2. Separate DNA From Crude Lysate
• DNA must be separated from proteins and
cellular debris.
Separation Methods
a) Organic extraction
b) Salting out
 a) Separation by Organic Extraction
 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.
 Phenol: Denatures proteins and solubilizes denatured
proteins
 b) Separation by Salting Out

• At high salt concentration, proteins are dehydrated,

lose solubility and precipitate.
Usually sodium chloride, potassium acetate or
ammonium acetate are used.
• Precipitated proteins are removed by
centrifugation
• DNA remains in the supernatant.
 Separation by Salting Out
Salting out method:
Cell lysis.
Protein digestion by proteinase enzyme.
Protein precipitation by high salt concentration.
Centrifugation will remove the precipitated
proteins.
The supernatant contains the DNA.
DNA is then precipitated by adding ethanol.
The precipitated DNA is resuspended in the
desired buffer.
 Ethanol precipitation:

-Precipitation of DNA: Absolute Ethanol is layered on the top

of concentrated solution of DNA
- Fibers of DNA can be withdrawn with a glass rod
- Washing of DNA
- Desalt DNA: Most salts are soluble in 70% ethanol
 2- Use of Commercial DNA purification kits:

 The common lysis solutions contain

A. sodium chloride
B. Trimethamine (also known as tris ) , which is a buffer to retain
constant pH
C. Ethylendiaminetetraacetic (EDTA) , which binds metal ions
D. Sodium dodecyl sulfate (SDS) which is a detergent .
E. An enzyme used in DNA extraction is protienase K
3- Heat denaturation
Achieved by boiling samples.
Heating of a sample to 100 c releases DNA into the
solution but also denatures it by separating the two
strand.
Drawbacks: There are remaining inhibitors in the
form of degraded proteins and other organic
compound or ions .
4- 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.
 Summary of DNA extraction :
There are three basic & two optional steps in a DNA
extraction :
1- Cell lysis , to expose the DNA within .
2- removing membrane lipids by adding a detergents or
surfactants .
3- removing proteins by adding a protease .
4- removing RNA by adding an Rnase.
5- precipitating the DNA with alcohol- usually ice cold
ethanol. In these alcohols , DNA strand will aggregate
together, giving a pellet upon centrifugation . This step
also removes alcohol- soluble salt.
 DNA Extraction & 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
Spectrophotometers

Nanodrop Qubit® 3.0

Measurement of DNA integrity
Checking for Degradation DNA
 Running your sample through an agarose gel is a
common method for examining the extent of DNA
degradation. Good quality DNA should migrate as a high
molecular weight band, with little or no evidence of
smearing.
 DNA absorbs UV light at 260 &280 nm & aromatic
proteins absorb UV light at 280 nm Apure sample of
DNA has the 260/280 ratio at 1.8 & is relatively free
from protein contamination.
 A DNA preparation that is contaminated with protein
will have a 260/280 ratio lower than 1.8
Checking the Quality of your
DNA by gel
The product of your DNA extraction will be
used in subsequent experiments
Poor quality DNA will not perform well in PCR
You will want to assess the quality of your
DNA extraction using the following simple
protocol:
 Mix 10 µL of DNA with 10 µL of 2x DNA loading buffer
 Load this mixture into a 1% agarose gel
 Analyze results (the following slides provide guidance)
 Agarose gel electrophoresis
Principles of nucleic acid separation by agarose gel electrophoresis

Agarose gel electrophoresis is a routinely used method for separating

proteins, DNA orRNA. (Kryndushkin et al., 2003).

Nucleic acid molecules are size separated by the aid of an electric field
where negatively charged molecules migrate toward anode (positive)
pole.

The migration flow is determined solely by the molecular weight where

small weight molecules migrate faster than larger ones (Sambrook &
Russel 2001).

In addition to size separation, nucleic acid fractionation using agarose gel

electrophoresis can be an initial step for further purification of a band of
interest.
Agarose gel concentration
 Electrophoresis buffer
Various buffers are used for agarose electrophoresis.

The two most common buffers for nucleic acids are :

1. Tris/Acetate/EDTA (TAE) and
2. Tris/Borate/EDTA (TBE).

DNA fragments migrate with different rates in these two

buffers due to differences in ionic strength.

Buffers not only establish an ideal pH, but provide ions to

support conductivity.
In general, the ideal buffer should
1. produce less heat,
2. have a long life and
3. a good conductivity.
 Agarose gel running voltage
• Voltage
Migration of fragments in an agarose gel depends on the
difference in electric current.

Different optimal voltages are required for different

fragment sizes. For instance, the best

resolution for fragments larger than 2 kb could be

obtained by applying no more than 5 volts per cm to the
gel
Agarose gel sample loading and
visualization
 Agarose gel sample visualization
Ethidium bromide is the common dye for nucleic acid visualization.

The early protocol that describes the usage of Ethidium bromide (2,7-
diamino-10-ethyl-9-phenylphenanthridiniumbromide-) for staining DNA
and RNA in agarose gels dates as far back as 1970s (Sharp et al.,
1973).

Although the with a lower efficiency compare to the double- stranded

DNA, EtBr is also used to stain single- stranded DNA or RNA.

Under UV illumination, the maximum excitation and fluorescence

emission of EtBr can be obtained from 500- 590 nm.

Exposing DNA to UV fluorescence should be performed rapidly

because
nucleic acids degrade by long exposures and thus, the sharpness of the
bands would be negatively affected.
Agarose gel sample visualization
Vertical electrophoresis
Gel Electrophoresis
Expected Results in a Research Lab
Below is an agarose gel that has 5 genomic DNA samples from various plants.
Note that the DNA runs at a very high molecular weight and as a clear, thick band.
This DNA was extracted in a research lab under optimal conditions

1 kbp and 100 bp Genomic DNA of 5

ladders species of cereals
 Analyzing DNA Samples
in a Research Lab
If properly done, genomic extraction should result in bright bands in
the very high base pair range of a gel electrophoresis.

Sizes of Genomic DNA for

various Species in kbp
E. Coli 4,640,000bp
Yeast 12,100,000bp
Fruit Fly 140,000,000bp
Human 3,000,000,000bp
Pea 4,800,000,000bp
Wheat 17,000,000,000bp

The genomic fragments run at ~12kbp because they are sheared during extraction
 Expected Results in a Classroom Lab
• This is expected. Even though this A B C D E
genomic DNA preparation is not perfect, it Ladder
is suitable for use as a PCR template
• Lane A: Barley
Lane B: Corn
• Lane C: Oat
• Lane D: Rice
• Lane E: Wheat

• Note that the DNA has sheared (particularly for wheat) – broken up into numerous
fragments and is not a clean single band at the top – these are the mid-ranged sized
fragments (1000-10,000bp size range)
• The bright bands at the 100 - 1000 bp range are RNA, which also gets extracted using
this protocol
 Analyzing DNA samples
in a Classroom Lab

Ladder
A B C D E
Analysis of samples:

Barley (A): This sample is fine

Corn (B): This sample is fine

Oat (C) : This sample is fine

Rice (D) : This sample is fine

Wheat (E): This sample has severe

degradation, can work for PCR
but should re-extract
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