Molecular Biology

(MLMB-201)

Lecturer:Dr. Mohamed Salah El-Din

Department of Medical Laboratory TechnologyFaculty of Allied Medical Science

Intended Learning Outcomes (ILO’s):

Molecular biology course provides an overview of the molecular basis to cell structure and function.This course focuses on the structure, biosynthesis and function of DNA and RNA on the molecular level and how these interact among themselves and with proteins. Molecular biology techniques are essential for modern biological and medical research. This course will give you an introduction to DNA and RNA standard techniques.

Student will have basic knowledge of:

• Cell organization.• DNA structure and function.• DNA Extraction.• RNA structure and function.• RNA Extraction.• Gene expression and protein biosynthesis.• Agarose gel electrophoresis for DNA/RNA; and SDS-PAGE for protein.• Polymerase Chain Reaction (PCR) – Theory, Types, Application.• Gene library and screening• DNA sequencing

Central Dogma Of Molecular BiologyCentral Dogma Of Molecular BiologyChromosomesThread-like structures present within the nuclei of cells. Chemically, a chromosome consists of an extremely long chain of DNA along with a coating protein. The combination of DNA and protein is called chromatin. The complete array of chromosomes in an organism is termed the karyotype.

Prokaryotic cell Versus Eukaryotic cell

A bit of DNA which acts as a code A bit of DNA which acts as a code to control part of a cell's to control part of a cell's chemistry. Every human cell chemistry. Every human cell holds, within its nucleus, more holds, within its nucleus, more than 50,000 different genes. Each than 50,000 different genes. Each gene is located at a particular gene is located at a particular position along the chromosome, position along the chromosome, termed termed locuslocus.. The particular form The particular form of the gene is termed the of the gene is termed the alleleallele..

What is a “What is a “genegene”?”?

DNA STRUCTUREDNA STRUCTURE

RNA DNA

Structure of

Nucleotide

Cytosine (C)

Cytosine (C)

Adenine (A)

Adenine (A)

Thymine (T) Thymine (T)

Guanine (G)

Guanine (G)

Deoxyribose)Sugar molecule(

Phosphoric Acid)Phosphate molecule(

Pyrimidine (T & C)Pyrimidine (T & C) Purine (A & G)

nucleoside nucleotide

Watson-Crick model of a Watson-Crick model of a linear,linear, double-stranded helixdouble-stranded helix

A single polynucleotide strand of DNA is a copolymer in which A single polynucleotide strand of DNA is a copolymer in which deoxyribose and phosphate alternate; the side chains are single deoxyribose and phosphate alternate; the side chains are single

organic bases, one attached to each deoxyribose.organic bases, one attached to each deoxyribose.

DNA FUNCTIONDNA FUNCTION

DNA is the repository of genetic information in all organisms:DNA is the repository of genetic information in all organisms:(except for a relatively small number of viruses)(except for a relatively small number of viruses)

It must transmit its encoded information to the cell which It must transmit its encoded information to the cell which contains it contains it “Transcription”“Transcription”

It must replicate in order that a progeny cell has the same It must replicate in order that a progeny cell has the same properties as its parent properties as its parent “Replication”“Replication”

Evolutionary pressures have produced a mechanism for Evolutionary pressures have produced a mechanism for creating new informationcreating new information “Mutation”“Mutation” and for exchanging and for exchanging genes genes “Recombination”“Recombination”, so that new and better organisms , so that new and better organisms can develop.can develop.

DNA ReplicationDNA Replication

MutationMutation

The copying process is not perfect, and very occasionally a fault occurs The copying process is not perfect, and very occasionally a fault occurs leading to a leading to a mutationmutation (change) in the nucleic acid sequence; this, in turn, (change) in the nucleic acid sequence; this, in turn, alters the structure of the DNA in one of the daughter cells-and thus leads alters the structure of the DNA in one of the daughter cells-and thus leads to a change in one of its genes. to a change in one of its genes.

MutationsMutations may potentially occur at any site within the genome and may may potentially occur at any site within the genome and may have several effects upon the structure and function of the genome. have several effects upon the structure and function of the genome.

RecombinationRecombination

Relevance ofRelevance of DNADNA isolationisolation

Isolation of DNA is often the first step before further analysis:

•• DNA profilingDNA profiling

•• cloningcloning

•• disease diagnosisdisease diagnosis

•• DNA sequencingDNA sequencing

•• genetically modified organisms (GMO) -genetically modified organisms (GMO) -agriculture, pharmaceuticalagriculture, pharmaceutical

• Environmental testing, bioterrorismEnvironmental testing, bioterrorism

DNA extraction

Protocol Highlights:Protocol Highlights: DNA ExtractionDNA Extraction

•• Collect cells from whole blood, tissues…etc.Collect cells from whole blood, tissues…etc. (NB. Ample cell collection is critical for success)(NB. Ample cell collection is critical for success)

•• Add Lysis buffer to cells to break open cell and Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contentsnuclear membranes and release nuclear contents

•• Digest sample with protease to degrade proteinsDigest sample with protease to degrade proteins

•• Precipitate DNA with cold alcohol in high saltPrecipitate DNA with cold alcohol in high salt

Lysis Buffer:Lysis Buffer:

What is Lysis Buffer?What is Lysis Buffer?

• 50 mM Tris-HCI, pH 8.0• 50 mM Tris-HCI, pH 8.0• 1% SDS• 1% SDS

Tris bufferTris buffer to maintain the to maintain the pH of the solution at a pH of the solution at a level where DNA is stablelevel where DNA is stable

1% SDS1% SDS to break open to break open the cell and nuclear the cell and nuclear membranes, allowing the membranes, allowing the DNA to be released into DNA to be released into the solution (SDS also the solution (SDS also denatures and unfolds denatures and unfolds proteins, making them proteins, making them more susceptible to more susceptible to protease cleavage).protease cleavage).

O

S

O

O

O

-

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

SDS

Why Add Protease?Why Add Protease?

•• Protease is added to destroy nuclear proteins that bind DNA and Protease is added to destroy nuclear proteins that bind DNA and cytoplasmic enzymes that breakdown and destroy DNA.cytoplasmic enzymes that breakdown and destroy DNA.

•• Protease treatment increases the amount of intact DNA that is Protease treatment increases the amount of intact DNA that is extracted.extracted.

Adding SaltAdding Salt• • The protease solution already contains saltThe protease solution already contains salt

•• NaNa++ ions of NaCI bind to the phosphate groups of DNA ions of NaCI bind to the phosphate groups of DNA molecules, neutralizing the electric charge of the DNA molecules, neutralizing the electric charge of the DNA molecules.molecules.

•• The addition of NaCI allows the DNA molecules to come together The addition of NaCI allows the DNA molecules to come together instead of repelling each other, thus making it easier for DNA to instead of repelling each other, thus making it easier for DNA to precipitate out of solution when alcohol is added.precipitate out of solution when alcohol is added.

• Salts and buffers deactivate the enzymes that degrade DNA Salts and buffers deactivate the enzymes that degrade DNA when released and stabilize the DNA (acid vs. base).when released and stabilize the DNA (acid vs. base).

Adding Ice Cold Alcohol?Adding Ice Cold Alcohol?

•• DNA does not dissolve in alcohol.DNA does not dissolve in alcohol.

•• The addition of cold alcohol makes the DNA clump together The addition of cold alcohol makes the DNA clump together and precipitate out of solution.and precipitate out of solution.

• Precipitated DNA molecules appear as long pieces of fluffy, Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands.stringy, web-like strands.

• Microscopic oxygen bubbles “aggregate” , or “fuse” together, Microscopic oxygen bubbles “aggregate” , or “fuse” together, as the DNA precipitates.as the DNA precipitates.

• The larger, visible air bubbles “lift” the DNA out of solution, The larger, visible air bubbles “lift” the DNA out of solution, from the aqueous into the organic phase.from the aqueous into the organic phase.

DNA extraction DNADNA is is easyeasy to prepare and store because to prepare and store because DNAasesDNAases

are easy destroyed by heating.are easy destroyed by heating.

1)1) Phenol/chloroform/isoamyl alcohol Phenol/chloroform/isoamyl alcohol extraction:extraction:Phenol is Phenol is organic solvent.organic solvent.

Nucleic acids prefer aqueous solutions.Nucleic acids prefer aqueous solutions.

But, proteins, lipids and carbohydrates prefer But, proteins, lipids and carbohydrates prefer organic environment.organic environment.

So, after addition of phenol to the cell lysate, the So, after addition of phenol to the cell lysate, the reaction tube will have two phases:reaction tube will have two phases:

o Upper aqueous layer containing DNA.Upper aqueous layer containing DNA.o Lower organic layer containing phenol + proteins.Lower organic layer containing phenol + proteins.

Phase separation by phenol extraction

2)2) Salting out method:Salting out method:Cell lysis.Cell lysis.

Protein digestion by Protein digestion by proteinaseproteinase enzyme. enzyme.

Protein precipitationProtein precipitation by by high salthigh salt concentration. concentration.

Salt also reduces the repulsion of the negatively Salt also reduces the repulsion of the negatively charged DNA molecules.charged DNA molecules.

CentrifugationCentrifugation will remove the precipitated will remove the precipitated proteins.proteins.

The supernatant contains the DNA.The supernatant contains the DNA.

DNADNA is then is then precipitatedprecipitated by adding by adding ethanol.ethanol.

The precipitated DNA is resuspended in the The precipitated DNA is resuspended in the desired buffer.desired buffer.

3)3) Spin column:Spin column:Commercial DNA extraction kits are based on Commercial DNA extraction kits are based on selective adsorption of DNA to selective adsorption of DNA to silica gel silica gel membrane columnsmembrane columns or or glass fiber matrix glass fiber matrix columns.columns.

Binding of DNA to silica or glass fiber is Binding of DNA to silica or glass fiber is enhanced by high salt concentration.enhanced by high salt concentration.

The bound DNA will then be eluted by The bound DNA will then be eluted by elutionelution buffer with low salt concentration.buffer with low salt concentration.

Extraction by spin column

Alkaline lysis method:Alkaline lysis method:Cell lysis.Cell lysis.

Then, we add Then, we add sodium hydroxide.sodium hydroxide.

The alkaline pH will denaturate chromosomal DNA The alkaline pH will denaturate chromosomal DNA but not the plasmid DNA.but not the plasmid DNA.

Then we add Then we add sodium or potassium acetatesodium or potassium acetate that will that will neutralize pH.neutralize pH.

So, chromosomal DNA will renature and aggregate So, chromosomal DNA will renature and aggregate with proteins.with proteins.

By centrifugation,By centrifugation, proteins + chromosomal DNA will proteins + chromosomal DNA will precipitate while plasmid DNA will remain in the precipitate while plasmid DNA will remain in the supernatant.supernatant.

Plasmid extraction

Quantification of extracted nucleic acids is Quantification of extracted nucleic acids is done by using spectrophotometer as follows:done by using spectrophotometer as follows:

At wave length 260: optical density (OD) of 1 At wave length 260: optical density (OD) of 1 means that:means that:

o The concentration of DNA= 50 µg/mlThe concentration of DNA= 50 µg/mlo The concentration of RNA= 40 µg/mlThe concentration of RNA= 40 µg/ml

So, the concentration of extracted DNA in a sample=So, the concentration of extracted DNA in a sample=

OD at 260 OD at 260 x 50 x dilution factorx 50 x dilution factor

Quantification

DETERMINING DNA CONCENTRATION AND PURITY BY SPECTROPHOTOMETRY

PROCEDURE: 

1. Fill two cuvettes with TE buffer. Read and record the A260 of the sample cuvette against the blank. Repeat at 280 nm.

 

2. Dilute the DNA in 400 µl of TE such that the A260 is ideally between 0.1 to 1.0. Mix well.

 

3. Empty and clean the sample cuvette and add the diluted DNA. 

4. Record absorbance of DNA sample at both 260 and 280 nm. Correct the readings as necessary using the blank values you determined in step 1.

 

5. The absorbance at 260 nm allows calculation of the concentration of DNA or RNA in the sample. An OD of 1 corresponds to approximately 50 ng/µl for double-stranded DNA, 40 ng/µl for RNA, and 32 to 34 ng/µl for single-stranded DNA and typical oligonucleotides. The A260/A280 ratio can provide a very rough estimate of the purity of the nucleic acid. Relatively pure preparations of DNA and RNA have A260/A280 values of 1.8 and 2.0, respectively. Phenol contamination will result in significantly lower A260/A280 ratios. Such contamination makes accurate quantitation of DNA or RNA impossible. Note however that the A260/A280 ratio can not be used to determine whether there is significant protein contamination in a nucleic acid preparation (Glasel, J. Biotechniques 18:62 (1995).

Spectrophotometer

It is very important to assess purity of the It is very important to assess purity of the extracted DNA (the degree of protein extracted DNA (the degree of protein contamination).contamination).

Purity is assessed by:Purity is assessed by:

Determine the ratio between the OD of the Determine the ratio between the OD of the sample at 260 nm and 280 nm. (OD 260/OD sample at 260 nm and 280 nm. (OD 260/OD 280).280).

If the ratio is 2: If the ratio is 2: this means that protein this means that protein contamination is zero.contamination is zero.

If the ratio is < 2:If the ratio is < 2: this means protein this means protein contamination of the extracted DNA.contamination of the extracted DNA.

N.B. Notice that the concentration of the extracted nucleic acid in a given sample can be roughly estimated by observing the fluorescence intensity of the band obtained on agarose gel after electrophoresis.

Congratulations!Congratulations!You have just created your You have just created your very own very own DNA Necklace!DNA Necklace!

The The DNADNA in the glass in the glass vial can last vial can last

for years. Add more for years. Add more alcohol into the vial if alcohol into the vial if

some evaporation some evaporation occurs.occurs.

Now You CanNow You CanCapture Your Unique Essence!Capture Your Unique Essence!

&&Prepare DNA NecklacesPrepare DNA Necklaces

Assignment:

As a part of the semester activity, one student is selected every week to prepare a short seminar about his/her point of interest in one of the lecture topics. That to be discussed and evaluated during the next lecture.