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Saturday, December 7, 2019

Endocytosis - Types of Endocytosis - Pinocytosis, Phagocytosis, Receptor Mediated Endocytosis


Endocytosis:

It is a process by which cells cab take in material from the extracellular environment. Endocytosis involves the cell membrane invaginating and engulfing the material into the cell. The intake of bulk material from surrounding into the cell through a membrane bound vesicle.

There are 3 different types of endocytosis:

  1. Pinocytosis
  2. Phagocytosis
  3. Receptor Mediated Endocytosis


Pinocytosis:



This is also known as cell-drinking. The cell uses the invagination to take in the surrounding bulk liquid or the aqueous media along with the dissolved solutes through the membrane bound vesicle into the cell. The vesicles of pinocytosis is relatively small.

Through the process of pinocytosis, cell gets nutrients and cell signalling components.

Liver cells, Kidney cells and epithelial cells most commonly uses pinocytosis.

Phagocytosis:

This is also known as cell eating. Cell uses pseudopodia to engulf large materials. These include bacteria, dead cells, etc.



Psuedopodia brings in engulfed material through membrane bound vesicles. The vesicles in phagocytosis are large compared to the vesicles of pinocytosis.
Phagocytosis process is very common to different cells of the immune system, these are macrophages, dendritic cells, neutrophils, etc. Once the bacteria is engulfed, immune system then process these vesicles.

Receptor mediated Endocytosis:



This is the most specific type of endocytotic process. It involves the ingestion of macromolecules such as sugars and hormones bind to receptor proteins on the cell membrane. This binding doe not only signal invagination but also the formation of protein coat around the vesicle. The vesicle is known as clathrin coated vesicle.

The main purpose is to ingest the macromolecules that bind to the receptor.

Sunday, October 20, 2019

Lac Operon : Gene Regulation in Ecoli (Prokaryotes)

Operons are groups of genes clustered in the bacterial DNA regulating the expression of functionally related proteins.

 In E. coli half of the genes are clustered into operons which are involved in particular metabolic pathways.

Lac operon model was proposed by Jacob & Monad.

Lac Operon is responsible for the transport and metabolism of lactose.

Tuesday, October 15, 2019

Taq DNA polymerase and its key features

Taq DNA polymerase is one the important component in the PCR Mastermix. Role of Taq DNA polymerase is to amplify the DNA strands. Let’s look at the features of Taq DNA polymerase in detail:



  1. Specificity
  2. Fidelity
  3. Processivity / Speed
  4. Thermostability

Specificity

One of the major challenge in PCR is the specific amplification of target DNA. Nonspecific amplification occurs due to the extension of misprimed targets and primer dimer formation. This can impact overall sensitivity and specificity of PCR assay.

To avoid amplification of misprimed template or primer dimer one option is to setup the pcr reaction in cold condition or add Taq polymerase as a last component in the reaction mixture. This method will help in reducing extension of primer dimer, misprimed template to some extent.

As the Taq polymerase has activity even at low temperature (activity will be less compared to its optimal temperature) extension of mispriming can occur during the reaction setup. To prevent this Hot-Start Taq polymerase has been developed.

Hot Start Taq polymerase will be active only after an activation step, which is much higher than the reaction setup temperature, thereby preventing extension of primer dimer and misprimed template.

Fidelity

Fidelity is the proof reading activity of DNA polymerase. This feature is essential for correct replication of DNA (without error). Cloning and site directed mutagenesis requires replication of DNA without errors. High fidelity polymerases are engineered for this applications.

DNA polymerase has two activities:

  • 3’ – 5’ proof reading activity
  • 5’-3’ Polymerase activity
3’-5’ exonuclease activity is the one determines the fidelity. This is also known as the proof reading activity. The ability to correct misincorporated nucleotides.

Due to polymerase activity, Taq DNA polymerase is able to incorporate nucleotides to the primed template. During polymerization, misincorporated nucleotides will be corrected by proof reading activity of the taq polymerase.

Taq DNA polymerase’s fidelity is often expressed as the inverse of error rate. No of misincorporated nucleotides per total number of nucleotides polymerized.

Processivity

Processivity of the taq polymerase is its speed or nucleotide incorporation rate per single binding event. High processivity Taq polymerases are required for amplifying longer targets in short time. It is beneficial in amplifying template DNA having higher GC content and also in amplifying crude samples, samples which are minimally processed.

New engineered versions of Taq polymerase are highly processive compared to the regular taq DNA polymerases.

Thermostability

PCR cycling involves repeated heating and cooling of the reaction mixture. Enzyme used in the reaction should withstand high temperature. Taq DNA polymerase isolated from bacteria living in hotsprings has revolutionized the PCR process.

Thermostability is the ability of taq DNA polymerase to withstand higher temperature. Taq DNA polymerase isolated from thermophilic bacteria is known for its thermostability. Half-life of the Taq polymerase reduces above 90°C.

The above features of Taq DNA polymerase makes it a versatile enzyme to be used in the polymerase chain reaction.

Monday, August 19, 2019

How to generate qPCR standard curve in excel - Tutorial

Generating qPCR Standard Curve in Excel & Calculating PCR Efficiency:

Plotting standard curve in excel from the qPCR Ct values and calculation of PCR efficiency from the DNA dilutions.

Saturday, June 29, 2019

Gel Extraction : Principle, Procedure & Tips for Improved Yield

What is Gel Extraction?

Gel Extraction or Gel Isolation is a technique used to excise and extract out the desired DNA fragment of interest after the agarose gel electrophoresis.

Why is it done?

Gel Extraction is used to purify the desired DNA fragment from unwanted materials, generally used to purify Restriction Digests, PCR Amplicons, etc.


Procedure:

Following steps are involved in the Gel Extraction:

  • Run DNA on Agarose Gel Electrophoresis, Excise the desired band after electrophoresis run.
  • Dissolve the DNA containing excised gel
  • Bind DNA to Silica Column
  • Wash the Bound DNA
  • Elute the DNA from column


Principle of Gel Extraction / Nucleic Acid Purification

Most of the commercially available kits uses spin columns for efficiently purify the DNA from agarose gel or from the aqueous solutions. Upto 10ug of the DNA can be easily purified with considerable recovery using the spin columns.

Guanidine HCL or Guanidine Isothiocyante is the widely used chaotropic salt.The adsorption of DNA to the spin column membrane occurs at higher chaotropic salt concentration, as the salt modifies the structure of water.

Adsorption of DNA to the membrane is also depended on pH. 95% adsorption at pH <7 .5.="" before="" column="" if="" is="" loading="" of="" ph="" solution="" spin="" the="" to="">7.5, a small amount of 3M sodium acetate pH 5.0 can be added to bring the pH below 7.5.

pH dependence of DNA adsorption to Spin Column membranes.

Elution:

Elution efficiency is depended on the salt concentration and the pH of the elution buffer. Elution efficiency is higher under basic condition and at low salt concentration. Low salt elution buffer 10mM Tris.Cl or Water can be used for elution. While using water for elution, pH should be with in 7.0 - 8.5.


DNA yield & Concentration:

DNA yield depends on these major factors:

1. Volume of Elution Buffer
2. The way elution buffer is applied to the column
3. Incubation of elution buffer on column


Tips Improving Yield of Gel Extraction:

  • TAE buffer is preferred over TBE if the gel extract is used for downstream enzymatic application.
  • DNA recovery may be higher in TAE compared to TBE.
  • Small DNA fragments give higher recovery compared to larger fragments.
  • Higher recovery is observed when binding step is done at slow centrifugation speed.
  • Incubating the elution buffer in column for upto 5 minutes can increase the yield.

Sunday, June 23, 2019

Dot Blot Technique: Principle, Procedure & Advantages

What is Dot Blot?

Dot Blot is a cheaper, easier and faster technique to detect the presence of Proteins and Nucleic Acids in a biological sample. Due to the simplicity of the technique it widely used as a ideal diagnostic tool.

This post covers the Protein Dot Blot Technique.

Dot Blot Principle

Dot Blot works based on the immunodetection principle for identifying specific protein.


Protein Dot Blot
Dot Blot - Principle
Dot Blot - Technique: Overview

Proteins are immobilized on to a protein binding membrane, PVDF or Nitrocellulose. After Protein Immobilization membrane is blocked to avoid non-specific binding, using blocking buffer.

Immobilized protein is then incubated with a primary antibody (for 30mins to 1 hour). The antibody is specific to the protein and is visualized either with a specific tag or with a secondary antibody. 

The secondary antibody generally will carry a tag that allow the visualization of protein.

Blots can be visualized using methods such as Chemiluminescence or Fluorescence.

Common Tags used in Dot Blots are enzymes conjugated to the secondary antibody. Upon addition of substrate, enzyme acts on the substrate to develop colour. Commonly preferred enzymes are HRP(Horse Radish Peroxidase) and Alkaline Phosphatase (AP)

Advantages of Dot Blot over Western Blot


Non-Electrophoresis Technique:
Dot Blot is non-electrophoresis technique, means that one need not do protein electrophoresis. Proteins can directly immobilized on to the membrane.

Simple & Easy Method:
No messy work of PAGE run and gel to membrane transfers, proteins are directly immobilized on membrane, making this method much easier and faster.

Time:
It is a faster method, takes ~3hours, whereas western blot may even take up to 2 days. Including the protein electrophoresis and gel to membrane transfer optimization.

Dot Blot is not as informative as Western Blot, but it is inexpesive, quick and easy method for the simple protein detection. 

Sunday, June 16, 2019

Plastids - Introduction, Classification & Functions

Plastids - Introduction:

Plastids are membrane bound organelle found in all plant cells and some protists (euglina). Dr Ernst Haeckel discovered plastid.

Plastid Classification:

Plastids are classified into two types, they are:

1. Leucoplast
2. Chromoplast



Plastid Classification
Plastid Classification


Leucoplasts:

  • These are colourless plastids (non pigmented).
  • Leucoplasts do not have grana.


    • Leucoplasts are found in Roots, Rhizomes, Tubers, etc. ,parts of the plants which are not exposed to sunlight. 
    • Main function of Leucoplasts are storage of food.
    Based on their function Leucoplasts are further classified into 3 Types:

    a. Amyloplasts - Stores carbohydrates. Location: Potato Tuber, Wheat, Rice, etc

    b. Allueronplasts - They are also known as proteoplasts. They stores aluerone protein. Location: Maize, Pulses.

    c. Elaioplasts - Stores Fat. These can be found in castor seed, ground nut, etc.


    Chromoplasts:

    • These are coloured plastids (Colored due to presence of Pigments).
    • Chromoplasts have grana / Thylakoid
    • Chromoplasts are found in Leaf, Petals, Fruits, etc. , parts of the plants exposed to sunlight.
    • Main function of chromoplasts are photosynthesis, attraction of pollinating agents.
    Chromoplasts are further classified based on the type of pigment present:

    a. Chloroplasts - Green Pigmented plastid, found in Green leaves, green stem,etc

    b. Pheoplast - contains Xantophyll & Fucoxanthin. These are found in brown algae.
     - 
    c. Rhodoplast - contains red pigment called to phycoerythrin. these are found in red algae.

    Some of the other pigments present in plants are:

    1. Lycopin - Red pigment found in Tomato.
    2. Capsanthin - Red pigment found in Red Pepper.
    3. Carotenes - Orange / Red pigment found in carrots.

    Leucoplasts and Chromoplasts are interchangable, means that when lecuoplasts attains pigments it becomes chromoplasts and when the chromoplasts lose pigments it can become leucoplasts.

    Sunday, March 17, 2019

    How to prevent PCR amplicon contamination

    PCR - Polymerase Chain Reaction is very sensitive technique for the amplification of Nucleic Acids. Due to its sensitive nature, billions of copies of DNA can be produced from tiny amount of initial DNA. If not properly handled this can lead to carry over contamination: from previously amplified PCR products.

    To eliminate the issue of carry over contamination, most common method used to remove amplicon / PCR product is to use dUTP instead of dTTP and the use of an enzyme in the reaction mixture called the UNG, Uracil DNA Glycosylase.

    Uracil DNA Glycosylase selectively removes dUTP incorporated PCR product,  without interfering with the target DNA amplification.

    Uracil DNA Glycosylase:

    Uracil DNA Glycosylase also known as UNG or UDG is an enzyme that degrades Uracil containing DNA.

    Uracil-DNA Glycosylase can be used to cleave DNA at any site where a deoxyuridylate residue has been incorporated. The resulting abasic sites can then be hydrolyzed by:
    1. Alkali treatment
    2. High temperatures
    3. Endonucleases that cleave specifically at abasic sites.
    Features of Uracil N Glycosylase
    1. The UNG enzyme is active only on Uracil containing DNA, inactive on RNA.
    2. Activity of the UNG enzyme is high on single stranded DNA(ssDNA) than double stranded DNA (dsDNA).
    3. Uracil DNA Glycosylase hydrolyzes the Uracil-glycosidic bonds at U-DNA sites in single stranded DNA and double stranded DNA, excising Uracil and creating alkali sensitive abasic sites in the DNA.
    Mechanism of Action of Uracil DNA Glycosylase



    Mechanism of Action of Uracil DNA Glycosylase

    Applications of Uracil DNA Glycosylase:

    • The enzyme Uracil DNA Glycosylase can be used in site directed mutagenesis experiments to increase the efficiency.
    • UNG can be used in highly labelled oligo nucleotide probe production.
    • Uracil-DNA Glycosylase can be used with dUTP to eliminate PCR carryover contamination from previous DNA synthesis reactions.
    Use of Uracil DNA Glycosylase for elimination of PCR product contamination:

    Uracil DNA Glycosylase enzyme is very specific to Uracil containing DNA, cleaves uracil-glycosidic bonds there by degrading the DNA.

    While setting up PCR reaction mixture, dUTP should be used instead of dTTP and UNG enzyme should be included in the PCR mixture.

    Checking UNG Enzyme functionality by conventional PCR:

    Generate PCR product using dUTP in the reaction mixture, Use a small amount of dUTP incorporated PCR product (10^5 fold diluted) as template for second round of PCR with UNG enzyme in the reaction mixture (follow intrusction guidelines by the manufacturer) and without UNG enzyme. Analyze the second round PCR product on Agarose gel.

    Checking UNG Enzyme functionality by Real Time PCR:

    Generate PCR product using dUTP in the reaction mixture, Use a small amount of dUTP incorporated PCR product (10^5 fold diluted) as template for second round of PCR with and ithout UNG enzyme in the reaction mixture (follow intrusction guidelines by the manufacturer).

    Analyze and compare Ct values in the reaction mixture with and without UNG enzyme.

    Types of Uracil DNA Glycosylase:

    Uracil DNA Glycosylase from Ecoli s widely used in PCR, but this is not completely inactivated at higher temperatures, whereas Uracil DNA Glycosylase from Atlantic Cod is heat liable and this is suitable for 1 step RT-PCR.

    Notes:
    Use of UNG prevents future contamination. Existing contamination of DNA amplified with dTTP cannot be removed.