AFLP Marker - Principle, Steps, Applications, Advantages & Disadvantages

 Markers are the tools that are used to distinguish DNA, individuals, populations or species. All the molecular marker techniques fall under two categories -
  1) The Restriction based
  2) PCR amplification based
The Example of restriction based technique is RFLP marker. In this technique the DNA is restricted with specific Restriction Endonucleases.
  PCR-based markers include SSRs, RAPDs, ILPs etc.

 In 1995 Peter Vos et al. developed a new marker type which is the combination of both the restriction based as well as the PCR based method. This marker type was named as AFLP or Amplified Fragment Length Polymorphism.

Principle of AFLP

   AFLP involves the digestion of genomic DNA using restriction endonucleuses, followed by adapter ligation and PCR amplification.
  The amplified products are visualised on high resolution polyacrylamide gels or automated sequencers. The variation in the length of fragments is analysed, which gives the estimate about genetic relationships among the individuals or the variations.

Steps of AFLP

1). DNA extraction and Restriction Digestion

AFLP technique requires very good quality of intake and pure genomic DNA, which should be free from protein and contaminants.
  The next step is the digestion with restriction endonucleuses. A rear cutter such as Eco R1 and a frequent cutter such as Mse-1 is used producing sticky ends.
- A rear cutter(Eco R1) identifies 6 base pair restriction site and cleaves it
- A frequent cutter(Mse-1) identifies and cleaves 4 base pair restriction site.

AFLP Marker Technique

2). Ligation of Oligonucleotide Adapters

  Adapters are double-stranded short oligonucleotide sequences of usually 14 to 20 base pairs. Two different adapters are used one each for Eco R1 and another for Mse 1.
  These adapters of known sequences serves as the target for PCR amplification.

3). PCR Amplificationtion

Amplification is done in two phases,
- The first phase is known as pre-selective amplification and
- The second phase is the selective amplification

a) Pre-selective amplification

It is the first round of amplification, in which few fragments are selectively amplified.
  A PCR reaction is set which contains
- The genomic DNA,
- dNTPs
- Polymerase
- Eco R1 and Mse 1 restriction enzymes
- Primers with one additional nucleotide
PCR of 20 cycles is set using the PCR product of pre-selective amplification step.

b). Selective Amplification

A second round of amplification is known as a selective amplification. In selective amplification more stringent primers (primer contain additional 3 nucleotides) are used to reduce the number of fragments amplified.
• Few cycles of Touchdown PCR is set to perform amplification.
In touchdown pcr the annealing temperature is lowered by certain degrees after every PCR cycle to, improve the amplification efficiency if AFLP.

4). Separation and Analysis

  the separation of fragments can be done on a 6% polyacrylamide gel or automated sequencer containing pop gels. the bending pattern of the fragments is analysed manually or with analytical software.
  Separation and analysis is to be performed on sequencer in that case fluorescently labelled primers are used during selective amplification.

Applications

• To detect various polymorphisms in different genomic regions.
• For identification of genetic variation in strains or closely related species of plants, fungi, animals and bacteria.
• AFLP technique has been used in criminal and paternity tests to determine slight differences within populations and in linkage studies to generate maps for QTL(Quantitative trait locus) analysis.

Advantages of AFLP marker

• As restriction sites are present across the whole genome of an individual which makes AFLP marker to analyse multiple locus at once.
• The sequence information about the organism is not essential as the primers complementary to the adapter sequences are designed.
• In contrast to RFLP which takes longer time for probe hybridization and more skills, AFLP is comparatively simple as PCR amplification of fragments is done
• AFLP is possible with lesser amount of genomic template
• The results are highly reproducible considering have a high quality of DNA as input.

Disadvantages of AFLP

• AFLP cannot be done with poor quality of DNA or degraded DNA
• As AFLP are dominant markers in nature they cannot detect homozygous or heterozygous individuals.
• One cannot ascertain which fragment belongs to which dna locus as AFLP are multi-locus in nature.

Summary

  AFLP is a highly sensitive method for detecting polymorphism in DNA.
  AFLP is a dominant type of molecular marker, it involves restriction followed by PCR amplification of genomic DNA.  
Primers and adapters are used for pre-selective and selective PCR amplifications.
  Fragments can be analysed either on polyacrylamide gel or automated sequencers. 

FISH (Fluorescent In Situ Hybridisation) - Defination, Principle, Steps and Applications

FISH (fluorescent in situ hybridisation) is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity.
  It is used to detect and localise the presence or absence of specific DNA sequences on chromosome.

Principle of FISH (fluorescent in situ hybridisation)

  FISH involves hybridising a fluorescent labelled DNA probe to denatured chromosomal DNA of metaphase chromosome, as well as I phase chromosomes.
   In FISH the DNA probe is either labelled directly by incorporation of fluorescent labelled nucleotides precursors or indirectly by incorporation of nucleotide precursors or containing a reporter molecule which after incorporation into DNA is then bound a pleasantly labelled affinity molecule.
   The position at which the probability to the chromosomal DNA is visualised by detecting the fluorescent signal emitted by the labelled DNA.

1). FISH analysis is performed by denaturing the double stranded DNA in fixed chromosome on a microscopic slide.

2). Once a denatured, two fluorescently labelled DNA probes are used to combination to analyse marker sequence location.

3). First probe serve as control and hybridize with DNA on target chromosome, but outside the target sequence.

4). The second probe hybridize  and locate the target sequence.
- If target sequence present, the probe will hybridize and fluorescence with colour other than control probe.
- If detection present, second probe will not hybridize and no fluorescent will seen in the fluorescent microscope.
- If the duplication two fluorescent spot will be seen with test probe.

Steps of FISH Assay

1). Preparation of Fluorescent Probes

- Fluorescent probes complementary sequences of target nucleic acid that are designed complementary to the sequence of inserts
- Size range from 20-40 base pair to 1000 bp.
- They are tagged with fluorescent dyes like biotin, fluracine and diaoxigenin.

2). Denaturation of Probe and the Target Sequence

- Denaturation is performed by heat or alkaline method on double stranded DNA in fixed chromosome on microscopic slide.

Hybridisation of Fluorescent
labelled probe with DNA

3). Hybridization of Probe and the Target Sequence

- Formation of duplex between two complementary nucleotide sequence
- It can be between DNA DNA, DNA RNA and RNA RNA.

4). Detection

Detection has Two types
a) direct labelling - lable is bound to the probe less sensitive
b) indirect labelling - require an additional step before detection.
Probe detected using antibodies conjugated to label like Alkaline Phosphate. It results into appreciation of signal.

5). Visualisation

- Fluorescent probe attaches to the target sequence during hybridization. This is visible light through a microscope with appropriate filters using fluorescent microscope.

Applications of FISH

•  FISH is used in germ cell or parental diagnosis of conditions such as aneuploidies.
• To detect and localised the presence and absence of the specific DNA sequence on chromosome and gene mapping.
• FISH also be used to detect and localise specific RNA target (mRNA and miRNA) in cells.
• Finding specific features in DNA for used in genetic counseling, medicine and species identification.
• Fish can be used to compare genomes of to biological species.
• Disease that are diagnosed using FISH include angelman syndrome, acute lymphoblastic leukaemia, cri-du-chat and down syndrome.
• The characterization of marker chromosome.
• Monitoring the success of bone marrow transplantation.

STS(Sequence Tagged Site) Mapping - Principle, Steps and Applications

  STS (Sequence Tagged Site) is a short sequence (200 to 500 base pair). That is unique in the genome. Its location and the sequence is known and therefore, it can be used as a landmark in genome mapping.

STS Mapping

   These STSs can be detected using amplification by PCR (Polymerase Chain Reaction) and are useful for localising mapping and sequence data.

  STS concept was given by olsen in 1999, in analysing the impact of PCR on human genome research. They found that single copy of DNA sequence of non map location could serve as a marker for genetic and physical mapping of gens along the chromosome.

EST (expressed sequence tag) :

  Sequence Tag Site derived from a region of DNA that is expressed in transcribed into mRNA is called EST.
  • Purified mRNA is used to generate the corresponding cDNA (Complementary DNA) by reverse transcriptase.
  • EST is the commonly used STS marker which is unique and produced by partial Sequencing of cDNA library clones.

  Since STS sequence may contain repetitive elements, sequence that appear elsewhere in the genome they posses market sequences at both ends of the site(STS) that are unique and conserved.

STS include markers as microsatellites -

  1. SSR : Simple Sequence Repeats
  2. STMs : Sequence Tag Microsatellite Sites
  3. SSRPs : Simple Sequence Repeat Polymorphism
  4. CAPs : Cleaved Amplified Polymorphic Sequence
  5. ISSRs : Inter Simple Sequence Repeats.

STS Mapping :

  A map representing the order and spacing of sequence tagged site, which in strength of DNA is known as STS mapping.
  STS mapping is essentially the same as map. Distances determined by linkage analysis except that the map distance is based on the frequency at which occurs between two markers.

Principle of STS Mapping :

  To Map STS or ESt sites, a collection of chromosome fragments is examined for the presence of each sts for EST. 
Fragments may be derived from a single chromosome as the whole genome.
   Neighbouring STSs will tend to be on the same fragment where as those very far apart will only rarely be found on the same fragment.

Steps in STS Content Mapping

1) Fragmentation of genome -  

 Fermentation of the genome into overlapping plants is usually done by using rare cutting enzymes (restriction enzyme) to produce large DNA fragments.

2). Preparation of large fragment library -

  The fragments does generated are ligated to clone vectors of higher capacity like YAC.

3). Overlapping clone identification -

Two strategies are used to identify overlapping clones.

a) Clone Fingerprinting -
  • Overlapping clones are identified based on common patterns of Restriction Enzyme fragments or repeat conserved between clones.
b) Chromosome walking -  
  • Overlapping clones are identified based on hybridization of two overlapping to a particular STS marker.

4) Preparation of SDS Content Map -

Arranging the overlapping fragments based on the presence or absence of a single particular probe.

Applications of STS mapping

  • STS content mapping is one of the best physical mapping methods by which high resolution is achieved.
  • STS based PCR produces a simple and reproducible pattern on agarose gel electrophoresis or polyacrylamide gel electrophoresis. In most case STS markers are Co-dominant i.e. allow heterozygotes to be distinguished from homeozygotes.

HAT Medium : Principle & Selection

 HAT stands for Hypoxanthine Aminopterin Thymidine. The Hypoxanthine and Thymidine are the bases which are converted into nucleotides by the enzymes HGPRTase and Thymidine kinase respectively.

  • The pathway of nucleotide synthesis, which involves these two key enzymes is known as the salvage pathway
  • Aminopterin is an inhibitor of the de novo pathway of nucleotide synthesis.

HAT Medium

  Mammalian cells can be isolated from their body. These cells can be grown in cell cultures that is under suitable artificial conditions.
  HAT medium is a selection medium for mammalian cell culture.
  Suppose we have two type of mammalian cells.
  1. First is the antibody producing. These cells have a limited lifespan in cell culture.
  2. Second cell type are immortal b-cells. These cells are capable of dividing indefinitely in cell culture.
  Another important thing to note is that these cells are mutant for the genes producing the enzyme HGPRT and antibodies. So we will represent these feature as HGPRT negative and IG negative.
 This means these cells cannot synthesize nucleotide by the salvage pathway.
They cannot produce their own antibodies.

We mix these two type of cells and expose them to polyethylene glycol(PEG). PEG is chemical fusogen.
In PEG medium the cells will fuse with each other. As a result of this fusion we will have five type of cells in the mixture -

  1. Unfused antibody producing b-cell
  2. Unfused immortal b-cell
  3. Fused antibody producing b-cells
  4. Fused immortal b-cells and
  5. Hybrid cells formed by fusion of antibody producing b-cell and immortal b-cell.

Five types of cells after fusion process
  

Separation of Hybrid cells

  Our next aim is to separate these hybrid cells from this mixture of cells. This cell mixture is now transferred to the HAT medium.

  • The antibody producing cells have a short lifespan in cell culture.
  • So after some time the unfused and fused antibody producing B-cells will die.
  • Immortal b-cells (HGPRT negative) cannot synthesize nucleotides by the salvage pathway.
  • Also this HAT medium contains Aminopterin which blocks the de novo pathway of nucleotide synthesis.
  • Since both the pathways of nucleotide synthesis are blocked. the unfused and fused immortal b-cells will also die.
  • The only remains is the hybrid cells formed by the fusion of antibody producing b-cell and immortal b-cell.
  • These hybrid cells will survive in the HAT medium. This is because they are able to divide indefinitely.
  • This property is due to the immortal B-cell partner.
  • Functional HGPRT enzyme for nucleotide synthesis is provided by the antibody producing b-cell partner.
So these cells will be able to synthesize nucleotides by the salvage pathway.

 HAT medium has the function of artificial selection in cell culture techniques. This medium selects those cells which have functional HGPRT and TK enzymes. 

 HAT medium is mainly used in Hybridoma Technology for production of monoclonal antibodies.

Basic Cell Cycle

  Each human begins his or her life from a single cell and this single cell is known as the zygote. Zygote divides again and again to form an infant further. it is the cell division because of cell cycle, we developed from an infant to an adult.
    Besides growth cell division is also involved in repairing our damaged tissues. This role of cell division is responsible for the healing of wounds cuts and injuries.

Cell Division
   For these functions of growth and repair, it is important that the
• New cells formed should be identical to the parent cells.
• New cells should be identical in size shape and functions.
•  The number of chromosomes in each daughter cell should be same as that of the parent cell.

This means for the production of genetically identical daughter cells, Each parent cell must first undergo a growth phase in which,
- it increases in size
- it duplicates its chromosomes and other important components necessary for cells function.

Cell cycle
   In eukaryotes cells undergo a repeating pattern of growth followed by division.
This repeating sequence of cellular growth and division during the life of an organism is known as the cell cycle.
  Eukaryotic cell it undergoes a growth phase, then it divides the genetically identical daughter cells produced may further enter into this cell cycle to produce more cells.
  Chromosome duplication also takes place before cell division. There are 2n chromosomes in the original cell these chromosomes get duplicated so now there are 4n chromatids. Finally after cell division each daughter cell has 2n chromosomes.

The cell cycle can be divided into two major stages
   1). Interphase and
   2). M phase

1). Interphase
• Cells actually spend most of their time in the interphase.
• In interphase the chromosomes uncoil into extremely long and thin structures. They cannot be distinguished as individual threads in this stage.
• Also under light microscopy no dramatic change is visible inside the cell.
• Interphase is a not resting stage of cell. Interphase is an active time.
•  The cell is fulfilling its specialized or routine functions in the individual's body.
• In terms of the cell cycle interphase is the stage during which a cell prepares itself for cell division.
• Interphase is longer stage accounting for 90% of the cell cycle.

interphase is further divided into three phases
  - The G1 phase
  - The S phase and
  - The G2 phase
•  G stands for gap, but these phases are not just gaps because cell growth takes place during these phases.
•  S stands for synthesis, during this phase DNA synthesis or chromosome duplication takes place.

The G1 Phase
   New cells arise from a pre-existing cells. G1 phase is the duration between the birth of a new cell and the onset of its chromosome replication.
• During G1 phase the cell grows rapidly and carries out its normal routine functions.
• Cytoplasmic organelles such as mitochondria and endoplasmic reticulum usually duplicate during this phase.

The S phase
  S stands for synthesis, more specifically synthesis of DNA during this phase.
• The cell duplicates its chromosomes. these are the n chromosomes before duplication, when these chromosomes are duplicated during S phase we get two exact copies of each chromosome.
• These copies of each chromosome are attached to each other at a point called centromere and also along the arms.
• Although the chromosome is duplicated but remains attached to its exact copy at the centromere. As long as they are joined together these duplicated chromosomes are considered as a single chromosome.
• These duplicated chromosomes are not visible during interphase.

The G2 phase
G2 phase is the time period between the end of S phase and the beginning of M phase.
• During this time cell further grows and synthesises proteins that are required in the subsequent cell division.
• An important event that takes place during this phase is the assembly of microtubules
• These microtubules are visible outside the nucleus.

These were the three phases of interphase. so all the preparation for cell division is complete. The cell now enters the next stage which is known as M phase.

M Phase
M phase or mitotic phase is the stage during which actual cell division takes place.
The main events of the stage are
- Chromosome segregation and
- The division of cytoplasmic contents.
• M phase includes two main processes -
1). Mitosis and
2). Cytokinesis

Mitosis is the process in which duplicated chromosomes are separated into two nuclei. Each of these two nuclei contains same number and kind of chromosomes as the original cell.
On the other hand Cytokinesis is the process in which the entire cell divides into two daughter cells.
• M phase is the shortest part of the cell cycle 

NATIVE PAGE - Principle



  PAGE stands for Poly Acrylamide Gel Electrophoresis. This technique is mainly used to analyse proteins and small fragments of nucleic acids. Here we will focus on separation of proteins using PAGE. As the name indicates Poly Acrylamide gel used in this method

why we use polyacrylamide gel instead of agarose gel ?
The answer to this question is hidden in the average pore size of gel matrix in each case. Agarose gel has larger pores as compared to polyacrylamide gel, which has smaller pores.

 
   Therefore agarose gel is suitable for electrophoresis of large molecules, such as DNA and poly acrylamide gel is suitable for the electrophoresis of smaller molecules such as proteins.

Various factors on the basis of which proteins get separated
  In polyacrylamide gel electrophoresis separation of proteins depend on their
- charge density (charge to mass ratio)
- size (or Molecular weight) and shape.

Charge density (charge to mass ratio) of the protein molecule
  Proteins are composed of amino acids. Each of these amino acids carries charge either positive or negative. Also some amino acids may have no charge.
   Thus because of these individual charges on the amino acid residues, the protein molecule will carry an overall or net charge.
   The Net charge carried by a protein molecule depends on pH of its surroundings. So the net charge on a protein molecule can be positive negative or neutral at a particular pH in gel electrophoresis.
   pH of the buffer is set such that all the protein molecules at that pH will carry a negative net charge. Being negatively charged these protein molecules will migrate to the positive electrode or anode.

  Suppose there are two protein molecules in a given sample.
The first one has higher charge density and the second one has lower charge density as compared to the first.
The protein molecule with greater charge to mass ratio or charge density migrates faster in the gel.

Size and Shape of Protein molecules
   Proteins are of different sizes and shapes. Size is due to the number of amino acid residues in the protein molecule. This also means larger the size higher will be its molecular weight.
   The shapes of protein molecules they exist in various forms, Such as globular, elongated etc. The shape of protein depends on their ability to form primary, secondary, tertiary and quaternary structures.
   The extent of cross-linking and the average pore size of the gel affects the migration of proteins of various shapes and sizes.
Size -
   As larger the protein molecule, slower will be its migration. This is because larger protein molecules become entangled in the molecular sieve formed by the gel.
Shape -
  Compact globular proteins migrate faster than elongated proteins of comparable molecular weights.

So in page migration of proteins depend on the combination of multiple factors these are
- charge density
- size and shape of the protein molecules.
Here, The protein molecules remain in their Native form or in other words they remain intact. This type of page in which intact proteins in a sample are separated is known as Native page.
  This method is preferred when our requirement is to detect a particular protein on the basis of its biological activity. For example
• Separation and detection of enzymes
  Protein electrophoresis using native page has also some limitations. For example native page is not suitable for determination of molecular weights of proteins. 

Pyrosequencing - Principle and Steps

  Pyrosequencing is a DNA sequencing method. In which incorporation of dNTPs in the DNA is detected in a form of lite.

Basic principle 

New DNA strand is synthesized on the template strand.
  During DNA synthesis a phosphodiester bond is formed between the last nucleotide of the growing strand and the incoming complementary nucleotide. As a result Pyrophosphate (PPi) is released.
   Thus every time a complementary nucleotide is incorporated in a strand being synthesized, a pyrophosphate will be released.
  This pyrophosphate is detected in pyrosequencing and forms the basis for the determination of the DNA sequence of the template strand.

Detection of released Pyrophosphate

  The  pyrophosphate is detected by an enzyme cascade reaction that results in the emission of light.
  The emission of light confirms that a pyrophosphate has been released. 

Reactions 1

  When released pyrophosphate combines with substrate known as Adenosine Phosphosulphate (APS) in the presence of an enzyme ATP sulfurylase, The ATP  is generated.
•  PPi + APS ➞ ATP + Sulfate (catalyzed by ATP-sulfurylase)

Reaction 2

In the next reaction this ATP is utilized by the enzyme luciferase for the conversion of Lucifer into Oxyluciferin and production of light.
• ATP + luciferin + O₂ ➞ AMP + PPi + oxyluciferin + CO₂ + Light (catalyzed by luciferase);
  Thus the pyrophosphate released during DNA synthesis can be detected by the emission of light. This detection of pyrophosphate is the basis of DNA sequencing and hence the name pyrosequencing.

Requirements for pyrosequencing

• A DNA fragment, That we want a sequence. This DNA fragment will be our template strand. This DNA fragment is engineered at one end with a sequence that is complementary to a primer. 

Primer

Deoxynucleotides dNTPs (dATPαS, dCTP, dGTP, dTTP)
  here the normal dATP is replaced by dATPαS. That is deoxyadenosine alpha Theotriphosphate.

  This replacement is necessary, this is because enzyme luciferase also uses ATP to produce light. To avoid false signals of pyrophosphate protection during pyrosequencing this replacement is done. dATPαS is used by DNA polymerase but not by luciferase.

• Enzyme DNA polymerase, for new strand synthesis.
• Other substrates required
  - Adenosine Phosphosulphate (APS)
- Luciferin
• Other enzymes required
  - ATP sulfurylase
  - Luciferase
  - An enzyme known as Apyrase also needed. This enzyme removes unused nucleotides. Thus it is a nucleotide degrading enzyme.

Steps involved in Pyrosequencing

Step 1 - Incubation of template strand with the primer

   The DNA fragment of unknown sequence is taken. This DNA sequence is engineered at one end that is complementary to a known primer.
  This DNA fragment serves as DNA template strand and it is incubated with the primer. The primer binds to its complementary sequence on the DNA template strand.

Step 2 - Addition of Enzymes and Substrate

In this step DNA polymerase is added, along with the other enzymes (ATP sulfurylase, Luciferase) and substrates (Adenosine Phosphosulphate, Luciferin) required for the detection of pyrophosphate.

Step 3 - Addition of One Type of Nucleotide

  After the second step one of the four types of nucleotides is added.
Here note that only one type of nucleotide is added at a time. That means if we are adding dCTP, then the solution of nucleotide we are adding contains only dCTP molecules.
   If the added nucleotide is incorporated in the new strand, pyrophosphate will be released and emission of light will take place. This light is detected by a detector and later used to interpret the unknown sequence the details of this we will see shortly.
But if the added nucleotide is Not incorporated, then there will be no pyrophosphate and therefore no light emission. This happens when the incoming nucleotide is not complementary to the nucleotide of the template strand.

Step 4 - Enzymes Apyrase is added for removes unused nucleotides

Now in both the cases whether the nucleotide is incorporated or not. Extra or unused nucleotide will be present.
  These extra nucleotides are now removed from the reaction. This is done by adding enzyme Apyrase the nucleotide degrading enzyme.

Step 5 - Reaction starts again with another nucleotide

  After the degradation of unused and extra nucleotides is completed. The pyrosequencing reaction starts again with the addition of next nucleotide.
  This process is repeated adding each nucleotide one after the other until the synthesis is complete. So every time a complementary nucleotide is added emission of light will take place.

Graphical Presentation

  The light emission in pyrosequencing is represented graphically to interpret the sequence. The Y-axis of graph represents the light intensity and the X-axis represents the sequence in which nucleotides are added.

Graphical presentation of pyrosequencing
 
  The peaks in the graph also give an idea about the number of same nucleotides present in the sequence. So as per the figure sequence of the Strand that is synthesized during pyrosequencing is C D A C D A G G G A. 
The sequence of the template strand will be complementary to this. Thus the sequence of the unknown DNA fragment is found using pyrosequencing. 

Factors Influencing Melting Temperature of DNA

Defination of Melting Temperature of DNA

  The melting temperature of the DNA is the temperature at which half of the DNA molecules are denatured.  

  That means at this temperature half of the DNA molecules present in the solution will be single-stranded and other half will be double-stranded. melting temperature is found at the midpoint of the melting curve besides denaturation.

Factors Affecting Melting Temperature

The melting temperature of DNA is affected by three main factors these are
- Nucleotide content of the DNA molecule
- Length of the DNA molecule and
- Ionic strength of the DNA solution

Nucleotide content of the DNA molecule

   DNA is double helix molecule. In DNA molecule Adenine always pairs with Thymine by 2 hydrogen bonds and Guanine always pair with Cytosine by 3 hydrogen bonds. Moreover the base stacking interactions in case of Guanine, Cytosine pair are more stable. 

   DNA molecule which is GC-rich in other words major percentage of this DNA molecule is made up of Guanine and Cytosine pairs and in the other DNA molecule in which Guanine, Cytosine pairs are less a DNA molecule which is GC-rich DNA will have higher melting temperature

  This is because more heat energy is required to disrupt the stable base stacking interaction in this molecule. Thus the melting temperature of DNA is influenced by it's GC content. This can also be shown graphically (figure).


As per the above figure, you can see two melting curves. As we know higher the GC content of DNA, higher will be the melting temperature. So the first DNA sequence which is GC-rich and the second DNA sequence is not GC rich. The melting temperature at first DNA sequence is higher than the second DNA sequence.

Length of the DNA molecule

  Second factor affecting the melting temperature of DNA is the lengths of the DNA molecule. 

   A longer molecule of double-stranded DNA requires more energy to get disrupted as compared to a shorter molecule. This is because longer the molecule greater the stabilizing forces between the two DNA strands more heat energy is required to dissociate the strands and hence higher will be the melting temperature.

Ionic strength of the DNA solution

  The third factor affecting melting temperature of DNA is the ionic strength of the DNA solution.

The backbone of a DNA helix is made up of sugar and Phosphate. Each phosphate group in a DNA strand carries a negative charge. Thus overall each strand of DNA molecule carries a negative charge. The negative charges on both DNA strands will repel each other. 

   In eukaryotic cells proteins known as histones play important role in compaction of DNA within the nucleus of the cell. Histone proteins are rich in basic amino acids their positive charge helped in neutralizing the negative charges on DNA molecule.

   In the laboratory the DNA molecules present in a solution are stabilized by adding positively charged ions, such as sodium (Na+). Being positively charged these ions bind to the sugar phosphate backbone and neutralize the negative charges on the phosphate groups. Thus DNA in a solution becomes stable ionic strength.

   Suppose we have same DNA molecules in the given two DNA solutions. But the sodium chloride added in the first solution is 50 milli molar and in the second solution sodium chloride added is 100 millimolar.


   This means ionic strength in second solution is more than the first solution. In other words more sodium ions are present in the second solution is compared to the first. 
  So in the second solution where ionic strength is high DNA molecule will be more stable and we will require more heat energy to denature these DNA molecules.
   But in the first solution since ionic strength is low DNA molecules will be less stable as compared to the first this is because negative charges, which are not mute relized by sodium ions will repel each other. 
  This will contribute in the disruption of forces between the two strands. Therefore less heat energy will be required to denature these DNA molecules.
   So we can conclude that higher the ionic strength of a DNA solution more heat energy required and higher will be the melting temperature similarly lower the ionic strength lower will be the melting temperature.

Southern blotting - Basic principle & Steps

 Blot refers to the membrane, on which biological molecules such as proteins and nucleic acids are absorbed or immobilised. The process of transferring these molecules from a gel to a membrane followed by their detection on the membrane is known as blotting

  When the macromolecule involved is DNA the technique is known as southern blotting. Southern is the last name of the scientist who first blotted DNA. He is Sir Edwin Mello southern.

  By analogy blotting involving RNA is known as northern blotting and for protein this technique is known as Western blotting. Both southern and northern blotting techniques are based on nucleic acid hybridization.

Southern Blotting

  Southern blotting has been used for the detection of a specific DNA fragment in a given mixture of DNA fragments or total cell.    

  Suppose we have isolated genomic DNA from bacteria. Now we want to find out whether gene or DNA sequence of interest is present in this bacterial genome or not. The researcher already knows the nucleotide composition of the target DNA sequence.

Step 1. DNA Digestion

  In the first step, the bacterial DNA is digested with a restriction enzyme. This step results in thousands of DNA fragments of various sizes. This is because the restriction enzyme cuts the chromosomal DNA at many different sites within the chromosome. 

  Again the researcher already knows that the target DNA sequence would be present within a specific restriction enzyme site. Therefore that restriction enzyme is used for the step of DNA digestion.

Southern blotting technique

Step 2. DNA Gel Electrophoresis

   The second step is the DNA gel electrophoresis. The separation of DNA fragments resulting from the first step is done by gel electrophoresis. The DNA fragments get separated according to their molecular weights.

   These separated DNA fragments are double-stranded but for hybridization step we require single-stranded DNA fragments. Therefore before moving to the next step these DNA fragments are denatured in the gel by exposure of Delta mild alkali(NaOH).

  The gel is soaked in a denaturing solution which contains sodium hydroxide. Sodium hydroxide denatures the DNA by disrupting hydrogen bonds between the two complementary strands.

Step 3. Blotting

   In the third step, bloating is done. This means the DNA fragments are transferred from the gel to a suitable membrane. This membrane can be nitrocellulose membrane or nylon membrane.

  Nowadays the most preferred membrane for such transfer of nucleic acids is nylon membrane. This is because it has a high tensile strength and better binding capacity for nucleic acids.

How this transfer is carried out ?

  There is the traditional way to transfer the DNA to the membrane. The basis of this transfer is the capillary action.  

System of southern blotting

  The tray is filled with the suitable transfer buffer. A support for gel on membrane is kept in this buffer. This is usually a glass plate an absorbent paper or blotting papers are placed on this support. These are placed such that they imbibe the buffer. They act as a wick

-  Next the gel containing DNA is placed on the top of it.
-  An nylon membrane of the same size as that of the gel is placed over it.
- Again a thick stack of blotting papers or absorbent paper is placed over the membrane.
- This is followed by three to four inches of paper towels and this complete stack of gel membrane blotting papers and paper towels are pressed down by putting a weight on top the buffer or liquid.

  From the tray now Rises through the gel taking with it the DNA molecules. Once the DNA molecules reach the nylon membrane they become adsorbed tightly to the membrane.

  The remaining liquid passes through the paper and is absorbed by the paper towels placed at the top. During this transfer the DNA fragments retain the same pattern of separation they had on the gel.  

   Another method to transfer DNA is by using the electric field. this method is similar to the Western blotting. The difference is that here we are transferring negatively charged DNA molecules from the gel to the membrane instead of proteins.

Step 4. Hybridization and Washing

    The next step is hybridization and washing. In this step the nylon membrane is now incubated with many copies of a nucleic acid probe. Probe is a labeled single-stranded DNA molecule. 

   Under appropriate conditions probe will bind to the target sequences pairing and form a double stranded DNA hybrid. So the probe hybridizes with the DNA fragment that is complementary to it. Unbound probes are removed by washing

Step 5. Detection

   In the fifth step detection of the bound probe is carried out. We find the location of the double stranded hybrid formed in the previous step. 

  The probe is detected by autoradiography, fluorescence or a color change depending on what label we have used in the probe.

Importance

  Southern blotting is useful for detecting major gene arrangments. This technique plays important role for example,
- In DNA fingerprinting,
- Identification of noval gene,
- Identification of structurally related genes in the species, etc.

Summary 

•  In southern blotting a mixture of DNA fragments is prepared by restriction digestion.
•  These DNA fragments are then separated by gel electrophoresis.
•  Next they are transferred and immobilised to a solid support such as nylon membrane.
•  Once immobilised the DNA fragments present on the membrane are now incubated with a specific probe.
•  The hybridization takes place between the probe and the DNA fragment that has complementary base pairs.
•  In the last step the location of the hybrid is detected by detection of the probe.

Denaturation(Melting Curve) and Renaturation of DNA


   DNA is a double-stranded molecule. It is stabilized by chemical interactions. Most important of these interactions is the hydrogen bonds.

   In DNA double helix structure adenine(A) always pairs with thymine(T) via 2 hydrogen bonds, cysteine(C) always pairs with guanine(G) via 3 hydrogen bonds.

   Inside living cells the two strands of DNA separate from each other during DNA replication. This separation disrupts the hydrogen bonds between the bases and this task is done by proteins called helicases.

    In the laboratory the hydrogen bonds of DNA double helix can be disrupted by two methods -
First method is by changing pH of the DNA solution and
second method is by heating the DNA solution.

Denaturation (melting)

   DNA solution is heated as a result hydrogen bonds are disrupted and the double-stranded DNA separates into single strands. This separation of DNA strands is known as Denaturation or melting. 

  Thus denaturation of DNA is the loss of helical structure of DNA as the temperature increases, the percentage of DNA denaturation also increases. This can be shown graphically and the curve obtained is known as the melting curve of DNA.


Measurement of DNA Denaturation

   DNA denaturation is measured by using spectrophotometer. This is based on the fact that all nucleotide bases consist of aromatic rings. These aromatic rings absorb light in the ultraviolet(UV) range. Nucleic acids such as DNA are made up of these bases, therefore DNA molecule will also absorb light in the UV range. 

  All bases of DNA that is Adenine, Guanine, Cytosine and Thymine have a strong absorbance at 260 nm. Double stranded DNA absorbs less light at 260 nm compared to single stranded DNA. This is because base stacking interactions in DNA double helix interferes with the absorbance. 

  So as DNA denatures are melts its absorbance increases. Thus absorbance of a DNA solution is measured under particular set of conditions. Sigmoid shape curve obtained by plotting the absorbance values with increase in temperature results in the melting curve of DNA.

Melting Curve of DNA
   Lower portion of the melting curve represents that all the DNA molecules in the given DNA solution are intact. That is they are double-stranded and, Upper portion of the melting curve represents that all the DNA molecules have completely denatured or melted. 

  So as per figure we can see that as temperature increases DNA molecule melt and the two strands separate. Also as DNA becomes single-stranded its absorbance increases. 

  The temperature at which half of the DNA molecules are denatured is called as the melting temperature of DNA. That means at this temperature half of the DNA molecules present in the solution will be single-stranded and other half will be double-stranded. melting temperature is found at the midpoint of the melting curve besides denaturation.

DNA Renaturation

  Another feature of DNA double helix is that the separated complementary strands of DNA can spontaneously reassociate to form a double helix. This happens when the temperature of DNA solution is lowered below its melting temperature this phenomenon is known as Renaturation or annealing. 

Polymerase Chain Reaction(PCR) : Basic Concept, 3 Steps, Types & Applications

  Polymerase chain reaction or PCR is an in vitro technique for generating large quantities of a specific DNA sequence. In simple words it is an automated version of DNA replication. A typical PCR reaction produces millions of copies of the amplified target DNA segment from the original DNA molecule.

  PCR technique was invented by Kary Mullis in 1985. He received Nobel Prize in Chemistry for the invention of PCR in the year 1993.

  Polymerase chain reaction the name it self tells us that in this technique DNA polymerase is used to produce copies of a target DNA sequence. It is a change reaction because the target DNA is repeatedly replicated as long as the procedure of this technique continues.

The Basic Principle behind the PCR :

   DNA double helix can be dissociated or melted by heating. This is known as denaturation. Once DNA strands are separated, it can be copied each of the two separated strands by using primers, deoxy nucleotides and DNA polymerase.

  Primer is allowed to bind to the separated strands. This is known as annealing. DNA polymerase carries out the process of DNA synthesis. This is known as extension.

  The result of this we will get duplicated DNA sequences. This process can be repeated many times and as a result we get many copies of the original DNA sequence.

Schematic representation of PCR reaction and replication of DNA

Some additional points about polymerase chain reaction

• PCR is a cyclic process.
• It consists of a series of 30 to 35 cycles.
• Each cycle has three steps :
   -  Denaturation
   -  Annealing and
   -  Extension
• Each cycle lasts from 3 to 5 minutes. This means we can have millions of copies of target DNA sequence in approximately 2.5 hours.

•  Polymerase chain reaction (PCR) takes place in small tubes made up of polypropylene. These tubes are known as PCR tubes.

• To automate PCR reaction these PCR tubes are kept in an instrument known as thermal cycler. It is also simply called as PCR machine.

• Thermal cycler is an automatic temperature control device. It automates cycling and incubation times for the reaction.
 

Requirements for PCR reaction

  PCR is an in-vitro DNA replication reaction so there are four basic components required for this reaction to take place.

1). Double-stranded DNA segment

  It is the source of the target DNA sequence that is to be copied in PCR. Both the strands of the DNA sequence act as template strands for DNA replication. One important point here is that we should have prior knowledge of the sequences at the border of the target gene segment. This is essential for our second requirement.

2). 2 Different Single-Stranded DNA primers

  Primers are short approximately 12 to 24 nucleotides long, chemically synthesized DNA sequences.

  One of these primers is complementary to the border sequence of one strand and second primer is complementary to the border sequence end of the other strand.

  Primers will bind to these DNA strands such that, their 3' prime end will point towards each other.

3). Four deoxynucleotide triphosphates (dNTPs)

(dATPs, dGTPs,dCTPs,dTTPs)
   These deoxy nucleotides will be used by DNA polymerases to synthesize new strands during the replication process.

4). Taq (DNA) polymerase

   The fourth requirement is the enzyme, which catalyze the DNA replication reaction. A heat-stable DNA polymerase. This is because PCR is carried out at higher temperatures where normal DNA polymerase enzymes will lose their structure and therefore stability and their function.

  The most often used DNA polymerase in PCR is called Taq polymerase. Taq polymerase is named after the thermophilic bacterial species Thermus aquaticus. This bacterium lives in hot springs at near boiling conditions.

Other more efficient polymerases for PCR are :
Pfu Polymerase (Pyrococcus furiosus)
Tli Polymerase (Thermococcus litoralis)
Tth Polymerase (Thermus thermophiles).

   All these reaction components are added in the PCR tube along with suitable buffer and then these tubes are kept in thermal cycler.

Steps of PCR reaction

1). Denaturation at 94-98°C

  The first step of PCR reaction is known as denaturation. In this step the reaction mixture is heated temperature during the step is about 94°C to 98°C for 45 to 50 seconds. It depends on the G+C content of DNA.

  At this temperature the double-stranded DNA in the mixture denatures into single strands.

2). Primer Annealing at 40-60°C

   The second step is known as primer annealing. sometimes also called as hybridization. In this step the temperature is slowly reduced. Temperature is about 40-60°C. At this temperature hybridization of primers to their complementary sequences on the DNA template strands takes place.

   In PCR both the separated DNA strands act as the template and we have two different DNA primers. So on first DNA template strand the primer binds complementary to the nucleotides at the border of the target sequence on the strand. Similarly other primer binds on the second DNA template strand. Note that three prime ends of both the primers are pointing towards each other.

3). Extension at 72°C

Third step is known as primer extension or simply extension. In this step the temperature is raised to about 70-75°C more specifically 72°C temperature is used. This temperature is optimum for the catalytic functioning of taq DNA polymerase.

  So in the third step Taq polymerase start extending the primer by copying the complimentary target DNA sequence. DNA synthesis is initiated at the 3' prime end of each primer and uses the separated DNA strands as a template.

  Afterer the completion of the first cycle the number of copies of the target sequence is doubled. We started with a single DNA sequence and now at the end of the first cycle, we have two copies of the DNA sequence. Here each of these two end products are made up of one original DNA strand and one newly synthesized long strand. 

Second Cycle of PCR

  Remember that for second cycle we have now two DNA double strands. That means after the denaturation step in the second cycle we will have four DNA template strands two of these are the original DNA stands and two are the newly synthesized strands.

  One of the two double-stranded DNA in the second cycle it undergoes denaturation annealing and extension again.  DNA sequence got doubled and we get four DNA copies.

  This cyclic process of DNA replication goes on doubling the DNA copies. With each subsequent cycle short template strands become more abundant. Because of this the target DNA sequence is successfully amplified.

Types of PCR

- Real-time PCR
- Quantitative real time PCR (Q-RT PCR)
- Reverse Transcriptase PCR (RT-PCR)
- Multiplex PCR
- Nested PCR
- Long-range PCR
- Single-cell PCR
- Fast-cycling PCR
- Methylation-specific PCR (MSP)
- Hot start PCR
- High-fidelity PCR
- In situ PCR
- Variable Number of Tandem
- Repeats (VNTR) PCR
- Asymmetric PCR
- Repetitive sequence-based PCR
- Overlap extension PCR
- Assemble PCR
- Intersequence-specific PCR(ISSR)
- Ligation-mediated PCR
- Methylation –specifin PCR
- Miniprimer PCR
- Solid phase PCR
- Touch down PCR, etc

Application of PCR :

PCR has limitless applications some important application are as of
- PCR include evolutionary studies - Forensic analysis such as for paternity testing, crime investigation
- Genome sequencing projects
- Disease diagnosis
- Agricultural testing etc.

Western blotting (Immunoblotting) : Working Principle, Steps & Analysis

Western blotting : Working Principle, Steps & Analysis


 Blot refers to the membrane on which biological molecules such as proteins and nucleic acids are immobilised. The process of transferring macromolecules from a gel to a membrane followed by their detection on the membrane is known as blotting. Western blotting is a widely used technique for the detection and analysis of proteins.

When the macromolecule involved is DNA the technique is known as Southern blotting. Southern is the last name of the scientist who first blotted DNA sir Edwin Melo southern. By analogy blotting involving RNA is known as northern blotting and for protein this technique is known as western blotting.

Western blotting is also known as immunoblotting, this is because antibody probes are used for the detection of the target protein on the membrane.
  There are five main steps involved in Western blotting these are
-  Protein gel electrophoresis
-  Protein transfer
-  Blocking
-  Antibody probing and
-  Detection

Step 1). Protein gel electrophoresis

The first step in Western blotting involves separation and characterization of proteins by Gel electrophoresis.

  Suppose we have three protein samples. Each of these samples contains thousands of proteins. Our aim is to detect presence of a specific protein in these samples. These protein samples are separated into their component proteins by gel electrophoresis.

  Most widely used technique for protein electrophoresis is sodium dodecyl sulfate polyacrylamide gel electrophoresis abbreviated as SDS page. This technique sets proteins according to their molecular weights.

   Another important point is that all the separated proteins in the gel have uniform negative charge. This is because of sodium dodecyl sulfate Which coats the proteins. So after the sds-page the band's represents proteins present in each sample. The left side of the gel is represent in the molecular weight markers.

Step 2). Protein transfer

   Once proteins have been separated by SDS-page, next step is to transfer these proteins from the gel to a suitable membrane. The membranes used in Western blotting are those having high affinity for proteins.

  They have excellent protein binding and retention capabilities. Most commonly used membranes in Western blotting are nitrocellulose and polyvinylidene difluoride abbreviated as PVDF. Protein transfer is done for further detection and analysis of separated proteins.

   The membranes are thinner than gels. Therefore when proteins bind to these membranes, their epitope saw binding sites are easily accessible to the antibodies.

Electrophoretic Transfer

  Method used to transfer proteins from gel to membrane is known as electrophoretic transfer.
In this method electric current is used to elude proteins from gel and transfer them to membranes. The gel and the membrane are placed in the electrophoresis chamber such that, gel is at the side of negative electrode and membrane is at the side of positive electrode.

   Proteins in the gel are negatively charged so they move out of the gel and migrate towards the positive electrode.

Transfer sandwhich

  For protein transfer a stack or transfer sandwich of gel and membrane is prepared first. This sandwich also consists of sponge and filter paper the arrangement of sandwich is like this " sponge, filter paper, gel, membrane, filter paper again followed by sponge".

Filter paper provides a uniform flow of transfer buffer through the gel. This facilitates the movement of proteins out of the gel and on to the membrane.
  The sponge maintained the proper pressure during the transfer.

  This sandwich of gel and membrane is held inside a non-conducting cassette, which keep gel on the membrane in close contact. This complete setup is kept entirely submerged under transfer buffer. Within a buffer tank the placement of cassette is such that gel is at the side of negative electrode, membrane is at the side of positive electrode.

   When electric current is applied, the negatively charged proteins move from gel and traveled toward the membrane. Which is at the side of positive electrode.

   One very important component of transfer buffer is methanol. Methanol helps to increase the binding of proteins to the membrane. This is because during the transfer of protein from gel to membrane methanol removes SDS from proteins.

  At the end of this step all the proteins from gel moved to the membrane and become tightly attached to it. so we have a membrane with copy of band pattern from gel.

Step 3). Blocking

   The third step is known as blocking. The membranes have a very high affinity for proteins our next step will be the addition of antibody to find out the presence of target protein.

   Antibodies are also proteins that means they can bind to empty spaces on the membrane, where no protein bands are present. This nonspecific binding of antibodies to the membrane is detrimental to the specificity and sensitivity of the assay. Therefore it is essential to block spaces that are not already occupied by a proteins. So in this step blocking agent is added to the membrane.

   Most common blocking agents used are bovine serum albumin and non-fat milk. These blocking agents will fill all the unoccupied sites on the membrane. Since these blocking agents specifically bind the membrane they will not disturb the already bound proteins on the membrane.

Step 4). Antibody Probing

   After the blocking step the membrane is incubated with primary antibody. Since this antibody is specific to our target protein it will bind to the protein on the membrane. After this excess of primary antibodies are removed by washing.

Step 5). Detection

   Final step of Western blotting is the detection followed by further analysis of the protein. The membrane is now incubated with labeled secondary antibody.

   Use of secondary antibodies are preferred to maximize the sensitivity of the detection. Multiple secondary antibodies can bind to the target primary antibody and this results in the amplification of detection signal.

  So when the membrane is incubated with labeled secondary antibody. These antibodies bind to the primary antibody that is already bound to the target protein on the membrane. Excessive secondary Antibodies are also removed by washing.

  For the secondary antibody label here is an enzyme. Most preferred enzyme for protein detection in Western blotting is horseradish peroxidase (HRP). 
  In the next step the presence of this enzyme is detected by adding a suitable substrate HRP acts on colorimetric or chemiluminescence substrates.

  The membrane is incubated with a colorimetric substrate such as 4-Cholor-1-napthol (4CN). The enzyme in the membrane catalyzes the oxidation of the substrate into an insoluble purple color product, and this purple color is visible on a blot.