Bio-Resource

Kinetics of antibody response: Primary and Secondary antibody response

noreply@blogger.com (Unknown) — Wed, 11 May 2022 16:00:00 +0000

Hemoglobin Structure Function & its Derivatives - Normal & Abnormal Hem...

noreply@blogger.com (Unknown) — Tue, 03 May 2022 16:56:00 +0000

Biosensors- Types and Applications

noreply@blogger.com (Unknown) — Sun, 02 Feb 2020 05:55:00 +0000

Biosensor is an analytical device containing an immobilized biological material, which can specifically interact with the analyte to produce a signal that can be measured.The biological material can enzyme, antibody, hormone, etc.

A biosensor has 2 components:

Biological Component: Enzyme, Hormone, Antibody, etc.
Physical Component: Transducer, Amplifier

Types of Biosensors:

Electrochemical Biosensors
Thermometric Biosensors
Optical Biosensors
Piezoelectric Biosensors
Immunobiosensors
Whole Cell Biosensors

Real Time PCR Controls

noreply@blogger.com (Unknown) — Sat, 18 Jan 2020 07:50:00 +0000

Real Time PCR - Primer Probe Design Consideration

noreply@blogger.com (Unknown) — Sat, 18 Jan 2020 03:09:00 +0000

Real Time PCR Analysis - qPCR Terms

noreply@blogger.com (Unknown) — Fri, 17 Jan 2020 17:56:00 +0000

Glycolysis

noreply@blogger.com (Unknown) — Sat, 04 Jan 2020 11:36:00 +0000

Glycolysis is derived from greek words Glyks and Lysis. Glyks = Sweet and Lysis = Splitting.

Glycolysis: Glyco = Glucose, Lysis = Breakdown.

Glycolysis is the metabolic pathway involves the splitting / breakdown of Glucose (6 carbon compound) systematically to two 3 carbon compound, Pyruvate. Glycolysis takes place in the cytosol of the cell.

Overview of Glycolysis

In Glycolysis, Glucose, which is a 6 carbon compound is broken down to 2 molecules of pyruvate, pyruvate is a 3 carbon compound. During this process energy is released in the form of ATP and NADH. The pyruvate generated from glycolysis enter other metabolic pathways to generate more energy.

The process of glycolysis can be explained in Two Phases: Phase 1 & Phase 2.

In Phase 1, Glucose is converted to two molecules of Glyceraldehyde 3 Phosphate, energy is utilized in this step as there is addition of phosphate.

In Phase 2, Glyceraldehyde - 3- phosphate is converted to Pyruvate.

Glycolysis

Step1: Glucose is converted to Glucose – 6 – Phosphate. This reaction is catalysed by the enzyme hexokinase. Kinases are the group of enzymes that catalyses phosphorylation. Hexokinase transfers phosphate from ATP to glucose converting it to Glucose 6 Phosphate. Since ATP is converted to ADP it is an energy utilizing reaction.

Step2: Glucose – 6- phosphate is converted to Fructose – 6- Phosphate. This is a simple rearrangement reaction. This is catalysed by the enzyme Isomerase.

Step3: Fructose – 6- Phosphate is converted to Fructose – 1, 6- bisphosphate. Addition of phosphate group to the 1^st carbon of fructose ring. The reaction is catalysed by phosphor Fructokinase. Since ATP is converted to ADP it is an energy utilizing reaction.

Step4 & 5: Fructose – 1, 6- bisphosphate is converted to glyceraldehyde 3 phosphate and dihydroxyacetone phosphate. This reaction is catalysed by the enzyme aldolase.

Glyceraldehyde 3 phosphate and dihydroxyacetone phosphates are isomers and are interconvertible by the enzyme isomerase. Since glyceraldehyde 3 phosphate enters the glycolysis pathway, the equilibrium of isomerization reaction is always towards glyceraldehyde 3 phosphate.

Every molecule of glucose is split into 2 molecules of pyruvate, so every reaction of second phase happens twice and produce twice the products.

Step6: Glyceraldehyde – 3 – phosphate is converted to 1, 3-bisphosphoglycerate. This reaction is catalysed by the enzyme glyceraldehyde 3 phosphate dehydrogenase. Phosphate group is added to the 1^st position of the phosphoglycerate. In this step NAD is reduced to NADH and which enters the electron transport chain to generate more energy. Also this step utilizes a molecule of inorganic phosphate.

Step7: 1, 3-bisphosphoglycerate is converted to 3 phosphoglycerate. This reaction is catalysed by the enzyme phosphoglycerate kinase. The kinase enzyme transfers the phosphate group at the first position of phosphoglycerate to adenosine di phosphate and an ATP molecule is generated in this process.

Step8: 3 phosphoglycerate is converted to 2 phosphoglycerate. This is a rearrangement reaction catalysed by phosphoglycerate mutase.

Step9: 2 phosphoglycerate is converted to Phosphoenol pyruvate. This reaction is catalysed by the enzyme enolase. Mg2+ is utilized in this reaction.

Step10: Phosphoenol pyruvate to Pyruvate. This reaction is catalysed by the enzyme pyruvate kinase. The phosphate group of the Phosphoenol pyruvate is transferred to ADP to generate a molecule of ATP.

Net Reaction of Glycolysis

1 molecule of 6 carbon glucose converted to 2 molecules of 3 carbon pyruvate.

2 ADP, 2 NAD+ and 2 Inorganic phosphates are utilized in net and 2 ATP & 2 NADH are produced.

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

noreply@blogger.com (Unknown) — Sat, 07 Dec 2019 11:38:00 +0000

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:

Pinocytosis
Phagocytosis
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.

Lac Operon : Gene Regulation in Ecoli (Prokaryotes)

noreply@blogger.com (Unknown) — Sun, 20 Oct 2019 16:17:00 +0000

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.

Taq DNA polymerase and its key features

noreply@blogger.com (Unknown) — Tue, 15 Oct 2019 14:44:00 +0000

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:

Specificity
Fidelity
Processivity / Speed
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.

How to generate qPCR standard curve in excel - Tutorial

noreply@blogger.com (Unknown) — Mon, 19 Aug 2019 17:14:00 +0000

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.

Gel Extraction : Principle, Procedure & Tips for Improved Yield

noreply@blogger.com (Unknown) — Sat, 29 Jun 2019 08:16:00 +0000

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.

Dot Blot Technique: Principle, Procedure & Advantages

noreply@blogger.com (Unknown) — Sun, 23 Jun 2019 12:13:00 +0000

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.

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.

Plastids - Introduction, Classification & Functions

noreply@blogger.com (Unknown) — Sun, 16 Jun 2019 07:51:00 +0000

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

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.

Know How the Nucleic Acid Extraction Works

noreply@blogger.com (Unknown) — Sun, 12 Mar 2017 15:05:00 +0000

Molecular biology work require DNA / RNA in the first place. Source of Nucleic Acid required may vary depend on the kind of work. Nucleic acid can be isolated from bacteria, Virus, Human, etc and most of the labs use Commercial Nucleic acid extraction Kits for Isolating DNA / RNA.

Here is how the Spin Column based Nucleic Extraction Kits work:

Sample source can be: Blood, Urine, Stool, CSF, Bacterial Culture, etc.

Urine sample for Nucleic Acid Extraction

Blood Sample for Nucleic Acid Extraction

There are 4 Main Steps in DNA / RNA Purification:

Lysis
Binding
Washing
Elution

Lysis:

Sample is lysed using Lysis Buffer. Lysis Buffer generally contains high concentration of Chaotropic Salts (Guanidine HCL, Guanidine Thiocyanite, Urea, etc). The role of chatropic salts is to destabilize Hydrogen bonds, vander wall forces and hydrophobic interaction. These salts destabilizes proteins like nucleases and also play key role in removing interaction of water from DNA.

Detergents and Proteases are also included in most of the kits, Detergents aid in protein solubilization and cell lysis, whereas Protease (Proteinase K widely used serine preotease in the nucleic acid extraction kits) helps in digesting proteins which may otherwise interfere in the downstream process.

Binding:

Once the sample is lysed, Next step is binding the DNA / RNA to the silica spin column. Chaotropic salts aided in Lysis also helps in binding nucleic acid to the column. To enhance the binding of nucleic acid to the column, Alcohol is used.

Spin Column

Alcohol can be Ethanol or Isopropanol (IPA). Percentage of Alcohol used may vary in different nucleic acid extraction kits. The amount of alcohol used has influence on the yield of nucleic acid.

Nucleic Acid Extractions kits are optimized amount of alcohol, which results in higher yields of the nucleic acid.

To the Lysed Blood sample, Ethanol is added and loaded on to the Spin column for binding.

Qiagen Blood Mini Kit Lysis and Binding Steps is as follows:

Pipet 20 μl QIAGEN Protease (or proteinase K) into the bottom of a 1.5 ml microcentrifuge tube.

Add 200 μl sample to the microcentrifuge tube. Use up to 200 μl whole blood, plasma, serum, buffy coat, or body fluids, or up to 5 x 106 lymphocytes in 200 μl PBS.

Add 200 μl Buffer AL to the sample. Mix by pulse-vortexing for 15 s.

Incubate at 56°C for 10 min.

Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.

Add 200 μl ethanol (96–100%) to the sample, and mix again by pulse-vortexing for 15 s. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.

The adsorption of Nucleic acid to the silica membrane is driven by dehydration and hydrogen bond formation, which competes against weak electrostatic repulsion. High concentration of chaotropic salt will help drive DNA adsorption and low concentration will release the DNA from the silica.

Washing

Once the Lysate is passed through column, DNA / RNA will stay adsorbed to the column and the proteins and other contaminants would have collected in the flow through. Even though most of the contaminants got removed, there is still lot of contaminants in the silica membrane along with the DNA, those contaminants needs to be first removed before eluting DNA from the Silica. This is achieved by washing the column multiple times.

If the sample used was blood, spin column will be tinted yellow colour, blood pigments and other protein contaminants needs to be removed from the silica.

Number wash steps used may vary depending on the extraction kit used. Generally a low chaotropic salt wash followed by ethanol wash is performed.

Nucleic Acid Extraction - Spin Column

Elution

The last step in the nucleic acid extraction process is elution. In this step the adsorbed DNA from the silica is released out using a low salt buffer or nuclease free water.

After the ethanol wash, a dry spin is done to remove residual ethanol content.

10mM Tris- EDTA or Nuclease free water is used as Eluant. pH of the elution buffer is generally 8 - 9.

To maximize the DNA yield, elution buffer is allowed stand on the silica for 1 min, before centrifuging.

To increase the concentration of the DNA, elution volume can be reduced. Elution volumes may vary depending on different kits.

Commercially Available Nucleic Acid Extraction Kits:

Qiagen - Nucleic Acid Extraction Kit

Promega Genomic DNA Extraction Kit

QIAamp DNA Mini Blood Mini Kit - Qiagen

NucleoSpin® Blood - Macherey Nagel

GeneJET Whole Genomic DNA Extraction Kit - Thermo Scientific

Wizard Genomic DNA Extraction Kit - Promega

Membrane Vesicular Traffic

noreply@blogger.com (Unknown) — Mon, 27 Feb 2017 15:21:00 +0000

There are various small vesicles that transport proteins from one organelle to another. These vesicles bud from the membrane of particular parent organelle and fuse with the membrane of a particular "Target" (Destination) organelle.

They are critical for sorting of proteins newly made in the rough endoplasmic reticulum and also for proteins internalized from the cell surface.

There are three types of coated vesicles, each with a different type of protein coat and each formed by reversible polymerization of a distinct set of protein subunit under regulated conditions.

The Coated Vesicle Types are:

Clathrin
COP - I
COP - II

The above three coat proteins are mainly involved in the vesicular traffic

The coats depolymerize into their subunits, After formation of vesicles by membrane budding. The depolymerized coat subunits are re-used to form additional vesicles.

Clathrin mediates transfer of vesicles that bud from the trans-Golgi network and the plasma membrane and that then fuse with late endosomes.

COP I mediates retrograde transport from the trans- to the medial- to the cis-Golgi, as well as from the cis-Golgi/cis-Golgi network to the rough ER. It may also mediate forward transfer of vesicles from the rough ER to the cis-Golgi network

COP II mediates transfer of vesicles from the rough ER to the cis-Golgi/cis-Golgi network; in some cases COP II-coated vesicles fuse with each other to form larger “ER-to-Golgi intermediate compartments” that are transported along a microtubule and eventually fuse to form the cis-Golgi.

Clathrin

Typical clathrin coated vesicles are 50 - 100 nm in diameter. The membrane bound coat vesicle consists of fibrous protein clathrin. Clathrin molecules have three limbed shape and are called Transkelion. Each limb contains one clathrin heavy chain and one clathrin light chain.

Even in the absence of membrane vesicles, clathrin transkelions can polymerize to form the cage like structure found around the coated vesicles. when clathrin polymerizes it forms a polygonal lattice with an intrinsic curvature.

Reference

Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman

Cooper G M, The Cell: A Molecular Approach, ASM Press, USA

RNA Degradation - Mechanism

noreply@blogger.com (Unknown) — Sun, 02 Oct 2016 08:04:00 +0000

Klenow Fragment - Sturctural Analysis, Comaprison and Applications

noreply@blogger.com (Unknown) — Wed, 04 Nov 2015 15:20:00 +0000

What is Klenow Fragment??

Klenow Fragment is the proteolytic cleavage product of DNA Polymerase I.

Klenow Fragment is generated when DNA polymerase I is cleaved using protease subtilisin.

Klenow fragment retains 5’-3’ polymerase activity and 3’-5’ proof reading activity.

Structure of DNA Polymerase

DNA Polymerase has three domains:

1. Polymerase domain.

2. Proofreading domain.

3. Exonuclease domain.

Klenow Fragment has only 2 domains:

1. Polymerase domain

2. Proofreading domain

Klenow Fragment lacks 5'-3' exonuclease domain.

Structural Comparison of DNA polymerase and Klenow Fragment

Applications / Advantages of Klenow Fragment

DNA sequencing by the Sanger dideoxy method.
Fill-in of 5´ overhangs to form blunt ends.
Removal of 3´ overhangs to form blunt ends.
Second strand cDNA synthesis.
Second strand synthesis in mutagenesis protocols.

References

NEB Technical Resources
Technical Resources, PDB
Other Internet Sources

Base Excision Repair Mechanism

noreply@blogger.com (Unknown) — Sat, 16 May 2015 13:35:00 +0000

Base Excision Repair Mechanism, is a DNA repair mechanism, where in the altered bases of the DNA, is removed or replaced. In Base Excision repair, glycosylase recognizes and removes the damaged base by hydrolysing the glycosidic bond.

The resulting abasic sugar, is removed from the backbone by endonucleolytic action.

Endonucleolytic cleavage also removes a-purinc or a-pyrimidinic sugars that arise by spontaneous hydrolysis.
After the removal of damaged nucleotide, DNA polymerase and DNA ligase act upon the damaged strand to restore.

Cells have different DNA glycosylases, with different specificities. For example Uracil glycosylase which specifically removes the damaged uracil.

Lets see How Does Glycosylase Detects damaged base?

Glycosylases, diffuse through the minor groove of the DNA until a specific kind of lesion is detected. Glycosylases removes, the damaged base by a mechanism called Base Flipping.

The damaged base project out or flip out of the DNA double helix, glycosylase goes and binds to that base and removes it.

Glycosylase can selectively cleave the nucleotide without cleaving the DNA backbone. When the nucleotide is removed, a gap is created. To repair the strand with the gap, the strand need to be cut out and that short stretch removed need to be synthesized. The site at which the gap is present is called as the AP site. apurinic site or the apyrimidinic site .

Once the gap is recognized on the DNA strand, AP Endonuclease comes into the scene to nick the strand. This is because for the DNA Polymerase one to act on the strand, DNA need to be nicked then only DNA polymerase start its function, that’s the reason AP endonuclease acts on the damaged strand to create a nick.

DNA Polymerase has two activities, exonuclease activity and the polymerase activity. In this case, DNA polymerase makes use of three prime to five prime exonuclease activity, to chew off some the bases, and at the same time DNA polymerase, starts adding up the chewed off bases by its polymerase activity.

Once polymerase completes its action Ligase, seals the nick and completes the process.

p53 Structure and Function - Cell Cycle Regualtion & Apoptosis

noreply@blogger.com (Unknown) — Sun, 10 May 2015 12:20:00 +0000

P53 is a tumor suppressor gene, present in eukaryotic cell. Protein encoded by p53 regulates cell cycle and function as tumor suppression, hence it is also known as TP53 or tumor protein.

p53 is referred to as “the guardian of the genome”, as its role in conserving stability by preventing genome mutation. “53” denotes molecular mass of p53 as it runs as 53KDa band on SDS – PAGE.

DNA Melt Curve Prediction using uMelt

noreply@blogger.com (Unknown) — Mon, 02 Mar 2015 17:27:00 +0000

DNA Melt Curve Prediction Using uMelt

How to analyze Primer Sequence or Oligonucleotide sequence designed for PCR / qPCR

noreply@blogger.com (Unknown) — Mon, 02 Mar 2015 17:21:00 +0000

Once a primer is designed for PCR or qPCR, it is important to analyze its properties like melting temperature (Tm), GC content, etc for efficient performance in PCR. Using IDT oligoanalyzer tool one can vary parameters and and analyze the effect on the input primer.

Sample Sequence : GTA TCT GAT CGT CTT CAC TCC C

Transfection - Basics, Principle, Mechanism and Optimization of Mammalian Cell Transfection

noreply@blogger.com (Unknown) — Sun, 01 Feb 2015 16:52:00 +0000

Transfection is the process of introduction of foreign DNA into the nucleus of eukaryotic cell. The cells which has incorporated exogenous DNA are called transfectants.

There are two types of Transfection possible,

Transient and
Stable Transfection.

In transient Transfection, the foreign DNA will not get incorporated in to the host genome, but genes are expressed for limited period of time (24-96 hrs).

Stable transfectants will have the foreign DNA incorporated into the genome.

Here is the overview of transient Transfection protocol.

Transfection Methodologies:

Following are the methodologies that are used for transfection, each of the below strategies has its own advantages and disadvantages, so one need to choose the suitable method based on the application.

Reagent Based
Instrument Based
Viral Mediated

1. Transfection - Reagent Based

Reagent based methods are simple, The reagent used, neutralizes the negative charge and condenses the DNA for effective uptake.

As the cell membrane is negatively charged, -vely charged DNA molecules will be repelled from the cells, for the cells to take up DNA the net charge need to be positive, that is the reason why reagents are used. The main principle of using reagents is to give the DNA a net positive charge either by reagents binding to the DNA or by forming complexes with the DNA.

Calcium Phosphate
Lipids
Cationic Polymers

Overview of Reagent Based Transfection

Calcium Phosphate mediated Transfection

The mechanism of DNA uptake by calcium phosphate mediated method is not well understood. In this method, the DNA to transfected is mixed with calcium chloride, then this is added slowly to a buffered saline or phosphate solution. Incubating this mixture with the cells to be transfected. The cells will take up the DNA by endocytic pathways,

Lipid Mediated Transfection - Lipofection

Polar lipids formed of highly positive charge head groups attached to hydrophobic tails.

Electrostatic Interaction between positive charges of the head groups of cationic lipids and negatively charged phosphate groups of the DNA are the main forces that allow DNA to be spontaneously associated with the cationic lipids.

Cationic Lipids used in Transfection

Examples of Lipids used in Transfection are:

DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)
DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane)
DOTAP (1,2-dioleoyl-3-trimethylammonium-propane)

Lipoplexes

Lipoplexes are organized lipid molecules in the form of micelle or liposomes, which when complexed with DNA can be used effectively for transfection.

The net positive charge of the lipoplexes are effective for transfection.

Reagents for Transfection are also made of helper lipids.

Helper lipids in conjunction with cationic lipids forms structures called liposomes, they can effectively encapsulate DNA.

Cationic Polymers mediated Transfection

Overview of Cationic Poymer mediated Transfection

Cationic polymers differ from cation lipids as they do not have hydrophobic moeity cationic polymers can more efficiently condense DNA.

There are 3 different types of cationic polymers used for transfection

Linear
Branched
Spherical

Cationic Polymers includes PEI and dendrimers.

PEI (Polyethyleneimine) – Very effective nucleic acid condensing agents.

Cationic Polymers like PEI forms complex with DNA called the Polypelex (DNA, Polycation).

This complex has net positive charge, are very effective in transfection.

DEAE - Dextran

DEAE dextran is a cationic polymer that tightly associated with negatively charged nucleic acid.

The Positively charge polymer:DNA complex comes into close association with negatively charged membrane.

Lipopolyplex

Lipopolyplexes are combination of lipids and cationic polymers which can form complex with DNA and aid in effective transfection.

Ideal Features of Transfection Complex

Size – 40 – 1000 nm.

Charge – Transfection complex should have a net cationic (+) charge.

Positive surface charge density – Zeta Potential (+mV).

Charge ratio:

N/P Ratio =

Reagent Concentration in Nitrogen residues (mM) / DNA Concentration in phosphate moeities (mM)

*N/P ratio is very important for optimization.

Transfection - Instrument Based

Nucleic acids are transported mechanically into the cells, following are the commonly used methods for instrument based transfection.

Electroporation
Biolistic Technology
Microinjection
Laserfection / Optoinjection

Electroporation

The procedure involves placing the cell suspension and the foreign material (eg: Plamsid) to be

transferred into plastic or glass cuvette.The cuvettes used here are specially meant for electroporation, they have aluminium electrodes at the sides. The voltage and capacitance is set and the cuvette inserted into the electroporator.

Once after electroporation, few ml medium is added , and is incubated at the the culture's optimal temperature for an hour or more to allow recovery of the cells.The transformed cells can be screened out using selectable markers in the plasmid (usaually antibiotic resisitance genes are used).

Advantages and Disadvantages of Electroporation

Electroporation has the following advantages and disadvantages

Advantages:

Versatility: Electroporation is effective with nearly all cell and species types.

Efficiency: A large majority of cells take in the target DNA or molecule.

Small Scale: The amount of DNA required is smaller as compared with other methods.

Disadvantages:

Cell Damage: If the electric pulses are given for long time or with more intensity, some pores may become too large or fail to close after membrane discharge causing cell damage or rupture
Nonspecific Transport: The transport of material into and out of the cell during the time of electropermeability is relatively nonspecific. This may result in an ion imbalance that could later lead to improper cell function and cell death.

Biolistic Transformation / Gene Gun

Biolistic Transformation is the transfer of nucleic acid into cells via high velocity nucleic acid coated microparticles

Laserfection/Optoinjection

This procedure uses laser light to transiently permeabilize cells in very short time. Various substances can be efficiently opoinjected including ions, small molecules, dextrans,plasmids, proteins, etc

Viral Mediated Transfection

Viral Vectors

Characteristics of Viral Vectors

Advantages and Disadvantages of different Transfection Methods

Evaluating Transfection Performance: Reporter systems

Green Fluorescent Protein
Luciferase Reporter
Beta – Galactosidase
Secreted Alkaline Phosphatase

Direct Visualization of Nucleic acid delivery

Nucelic Acid Labelling

Fluorescent labels – Cyanine Dyes, Fluorescein
Epitope Tags

Covalent linkage of dye to the DNA.

Factors affecting Transfection

Media
Nucleic Acid
Complexing Time
Cell Culture condition / Confluency
Harvesting Time

Transfection - Optimization

Media

Culture media plays a major role in transfection efficiency. Cell culture media mostly supplemented with serum. For efficient transfection transfection complex should be made in serum free media. The reason for serum free media usage for transfection complex formation is that, the serum has nucleases in it which can chew up the nucleic acids,

Antibiotics also should be avoided in the transfection complex formation media, as the antibiotics are charged, they can interfere in the transfection complex formation and there by reducing the transfection efficiency.

Other charged compounds like polyanions (eg: heparin sulfate) should be avoided and also surfactants like pluronic F68, mostly used as antifoaming agent, should be avoided.

Nucleic Acid

The quality of nucleic acid used for transfection is also important. The plasmid purity or the 260/280 ratio of the plasmid used for transfection should be greater than 1.8.

Most of the times plasmid preps gets contaminated with Endotoxins (endotoxins are toxic to cells),The endotoxin contaminated DNA inhibits the transfection or will yield very low transfection efficiency.

Complexing Time

Complexing time is yet another important factor which need to be optimized for better transfection efficiency. If the the complexing time is too then the complex formed will be big, which will be difficult for the cells to take up, there by reducing the transfection efficiency.

Ideal complex formation time is 15 - 30 mins, but this need to be experimented as the different cell lines will behave differently, complex formation time above 30 minutes will have low transfection efficiency.

For mRNA transfection ideal complex formation time will be 5 - 10 mins.

Cell Culture Conditions

Adherent cell confluency affects transfection.
Low confluency – 25% - Not good.
50 – 70% confluency is good for transfection.
At greater than 90% confluency, cells will not take up DNA to nuclei because of the dissociation of nuclear membrane.

References

Life Technologies , Technical Resources
Biorad, Technical Resources
Qiagen, Technical Resources
Mirus Bio, Technical Resoources

How does glucometer or Glucose monitoring device work?

noreply@blogger.com (Unknown) — Sat, 10 Jan 2015 08:58:00 +0000

Glucose testing is an important part of a diabetic’s daily health care. Without testing, a diabetic can easily become ill, because his glucose levels are not where they need to be.

To do glucose testing, a diabetic uses a glucose testing meter, which uses a glucose testing strip.

A glucose meter or glucometer, is a medical device used for measuring the approximate level of glucose in the blood.

Working principle of glucose meter:

The glucose meter, determines the concentration of glucose in the solution. Most glucose meters, are based on electrochemical technology, they use electrochemical test strips to perform the measurement.

A small drop of the solution to be tested, is placed on a disposable test strip, that the glucose meter uses for the glucose measurement.

Glucose meter test strips

Glucose strips, that are used for glucose monitoring from blood.

In each test strip, there is an enzyme called glucose oxidase. This enzyme reacts with the glucose, in the blood sample and creates an acid called gluconic acid.

The gluconic acid then reacts, with another chemical in the testing strip called ferricyanide. The ferricyanide and the gluconic acid, then combine to create ferrocyanide.

Once ferrocyanide has been created, the device runs an electronic current through the blood sample on the strip.

This current is then able to read, the ferrocyanide and determine how much glucose is in the sample of blood, on the testing strip. That number is then displayed on the screen of the glucose testing meter.

Methods used in electrochemical measurement of glucose

The two most common methods, used in electrochemical measurement of glucose are:

Colorimetric method and
Amperometric method.

Colorimetric method

In this method, the typical sensors such as LEDs or photo sensors form the analog interface. These sensors are followed by a Transimpedance Amplifier (TIA) for the glucose concentration measurement in the solution.

The Colour Reflectance principle is used in this method to sense the colour intensity in the reaction layer of the test strip by the photometry.

The glucose meter generates a numerical value, that is a measurement of the glucose concentration present in the solution.

Amperometric method

In this method, the electrochemical test strip contains a capillary that is used to draw in the solution placed at one end of the test strip. The test strip also contains an enzyme electrode containing a reagent such as Glucose Oxidase.

Glucose undergoes a chemical reaction in the presence of enzymes and electrons are produced during the chemical reaction. These electrons (i.e., the charge passing through the electrode) are measured and this is proportional to the concentration of glucose in the solution.

An ambient temperature measurement is also made in order to compensate for the effect of temperature on the rate of the reaction.

The solution sample (Blood) is placed on the test strip and the reaction of the glucose with the enzyme takes place.The flow of electrons will correspond to the flow of current through the working and the reference electrodes. This current will change according to the glucose concentration.

The current is measured using a transimpedance amplifier (current-to-voltage converter) and an analogue-to-digital converter (ADC). The output of the transimpedance amplifier will be seen as a variation in the voltage with varying glucose concentrations in the solution.

Factors affecting Glucose Measurement

Temperature,
Humidity,
Altitude, etc.

This is because the rate of the enzyme reaction depends on these and other factors.

References:

Glucose Meter Reference Design, Microchip.
Glucose Meter Fundamentals and Design, Freescale Semiconductor Inc.
Other Internet Sources

How does pH paper work?

noreply@blogger.com (Unknown) — Thu, 01 Jan 2015 16:08:00 +0000

what is pH?

pH is the negative logarithm of Hydrogen ion concentration.

pH = - log[H+]

Higher Hydrogen ion concentration indicates, acidic condition, and Higher Hydroxide ion concentration, indicates basic or alkaline condition.

When there is equal amount of hydrogen ions and hydroxide ions, it indicates neutral condition.

Why pH paper is used?

pH paper is used to determine if a solution is acidic, basic or neutral.

This is determined, by dipping part of the pH paper into a solution of interest, and watching the colour change.

The packages that pH paper comes in, often includes a colour-coded scale indicating the pH that, something has when the paper turns a certain colour.

For instance, if the paper turns a dark greenish-blue, the pH may likely be around 11 to 14.

If the paper turns red, the pH may likely be around 1 to 4.

How does pH Paper work?

The pH paper changes colour in different pH solutions is because of the chemical Flavin.

This molecule, which is an anthocyanin, is soluble in water and changes color in the presence of various types of solutions.

The pH paper is coated with this chemical, gives the property of changing colour at different pH solutions.

In the presence of an acidic solution, it turns red. In the presence of a basic solution, it turns greenish-Blue. In the presence of a neutral solution, it turns light green.