Basics

What Is A Pcr

What is a PCR?

The Polymerase chain reaction (PCR) technique, developed by Kary Mullis in 1984 is an extremely powerful method for gene amplification. It is used to produce multiple copies of nucleic acid region of interest and is the most widely used technique in molecular biology . The name, polymerase chain reaction, comes from the DNA polymerase enzyme which is used to amplify (replicate many times) a piece of DNA by invitro enzymatic replication. In PCR, even a single copy of DNA or RNA can be multiplied many folds thus making it easier to detect.
The PCR method basically involves preparation of the sample, the master mix and the primers, followed by detection and analysis of the reaction products. These steps are discussed below.

1) A DNA preparation containing the desired segment to be amplified also referred to as a target sequences usually obtained by isolation from plant or animal sources.

2) Two nucleotide primers (about 10-30 bases long) containing sequences complementary to the target sequence. A Primer is a short synthetic, oligonucleotide complementary to a section of the DNA which is to be amplified in the PCR reaction.

3) Four deoxynucleoside triphosphates, viz., TTP (thymidine triphosphate), cCTP (deoxycyctidine triphosphate), dATP (deoxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate).

4) A heat stable DNA polymerase e.g., Taq isolated from bacterium Thermus acquaticus, Pfu (from Pyrococcus furiosus) and Vent(from Thermococcus litoralis) polymerases.  
5) PCR Buffers: PCR buffer is necessary to create optimal conditions for activity of TaqDNA polymerase. Buffers often contain Tris-HCL, KCL, and sometimes MgCl2. 

At the start of PCR, the DNA from a which segment is to be amplified, primer molecules, four deoxyriboside triphosphates and the DNA polymerase along with appropriate quantities of MgCl2 are mixed together in a reaction tube and following steps are carried out sequentially.

 

PCR procedure involves three basic steps:

1) Denaturation : The reaction mixture is first heated to a temperature between 90-98°C (Commonly 94°C) that assures DNA denaturation by removing the hydrogen bonds between the complementary bases, thereby, yielding a single-stranded DNA molecule. This is the called denaturation step. The duration of this first step of the PCR cycle lasts for around 2 minutes at 94°C.

2) Annealing: In this second step of PCR cycle, the mixture is allowed to cool down at a general temperature of 40-60 °C that will permit the annealing of the primer to the complementary sequences in the DNA molecule. These complementary sequences are located at 3’ end of the two strands of the desired DNA segment. 

3) Primer Extension: In this final step of PCR cycle, the temperature in this step is now so adjusted that the DNA polymerase will synthesize the complementary strand using the 3’-OH of the primers. This reaction is usually carried out at 72°C for 2 – 5 minutes and is the ideal temperature for the polymerase enzyme to catalyze its function. 

TaqDNA polymerase extends the primers by using each single stranded (ss) target as a template for the construction of new complementary strands. The primers are extended towards each other so that the DNA segment lying between the two primers is copied. This results in duplication of both original DNA strands.The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified

The completion of the extension step completes the first cycle of amplification, each cycle may take around 4-5minutes and the extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. Usually 20-30 cycles are carried out in most PCR experiments. After PCR cycles, the amplified DNA segment is available in sufficient quantity  to be identified by size analysis using  gel electrophoresis in which the amplified DNA segment (amplicons) is compared with a known molecular weight marker called as DNA ladder that contains the DNA. The amplicons can also be cloned readily or used as reagent fragments of known size.
 

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PCR primer designing: 

The success of PCR depends on the use of correctly designed primers. A correctly designed pair of primers amplifies a single DNA fragment corresponding to the target region of the DNA molecule. Primers are so designed that they are exactly complementary to the template DNA. The primers used in PCR are between 20-30 nucleotides in length. The length of the primers is of great importance as it influences the rate at which primer molecules pair with the template DNA; this rate decreases as the primer length decreases. Therefore, if primers are too long, complete pairing of primers may not occur during the time allowed for annealing. In view of this, primers longer than 30 bases are rarely used.

Also, the primers must not have self-complementary regions as this would lead to hairpin formation with them and make them unsuitable for PCR. In addition, the two primers used in a PCR must not have regions complementary to each other as it would lead to primer-dimmer formation which is a product of duplex formation between the two primers. These may be then extended by DNA polymerase and are amplified and lead to competition with the PCR reagents there by resulting in the inhibition of the amplification of the DNA sequence targeted for PCR amplification. 
It is essential that the two primers used in a PCR reaction have identical melting temperature for the successful and specific amplification. In cases where the melting temperatures are lower than the annealing temperature, the primers may in these cases may fail to anneal and get extended. Also, a primer having melting temperature (Tm) higher than the annealing temperature may lead to mis-hybridization and extend at an incorrect location along the DNA sequence. The ideal annealing temperature must be low enough to enable hybridization between the template and primer and high enough to prevent amplification of non-target sites. The ideal annealing temperature is usually 5 °C lower than the melting temperature of the template-primer duplex.
The simple of rules for the design of PCR primers includes the following.

1) Primer length: Primer length plays an important role since annealing temperature depends on it. Ideally, a primer length should range from 18-30 bases in length. And also it’s better to avoid 4G’s or 4C’s at a stretch.

2) Primer composition: Base composition should be approximately 45-60% of guanine + cytosine.

3) Primers should not have secondary structures (e.g. Hairpin loops).

4) Ideally, primers should not contain sequences that are complementary to each other. 

5) Palindromic sequences should be avoided.

6) Optimum Primer melting temperature (Tm) between 52-58ºC is preferred which can be calculated using the formula.
Tm = 2(A+T) + 4(G+C)

7) Product position: Primer can be located near the 5' end, the 3' end or anywhere within specified length. However, it is well known that a 3’ terminal position avoids mis-priming..
The melting temperature (Tm) for a particular primer can be calculated using the formula.

Tm = [(no. of A + T) x 2ºC + (no. of G + C) x 4ºC].

Melting Temperature (Tm) is the temperature at which one half of the DNA duplex will dissociate and become single stranded. The concentration of primer in amplification reaction should be between 0.1 and 0.5 μM. Apart from manual inspection of various primer characteristics, a number of computer programs which are easy to work with are nowadays available that follow guidelines for primer selection.  

A commonly used method is Basic Local Alignment Search Tool (BLAST) search which enables in determination of primer binding regions and nucleotide sequences. BLAST, a algorithm, is a free NCBI tool  that combines Primer-BLAST primer design tool and BLAST search into one application and  searches similar regions between the sequences, compares with the already known nucleotide sequence and determines the statistical similarities between them.
Likewise, Smith-Waterman is a similar tool that helps in finding similar regions between two nucleotide or protein sequences.

Also, nowadays, a number of commercial software products such as called Beacon Designer, Primer Premier, Primer Select, DNASIS, FAST PCR, etc are available which enable DNA and protein analysis, secondary structure predictions, primer design, molecular modelling, development of cloning strategies, plasmid drawing or restriction enzyme analyses.

Variants of PCR: There are several variants of PCR such as: 

1) Real time PCR:  A variant of PCR that is based on detection and quantification of a fluorescent reporter. It enables the amplification and quantification of target DNA molecule at the same time as the reaction progresses. The quantification is measured by the amount of amplified product generated using fluorescent probes at each stage during the PCR cycle.

These probes are of two types:

A) DNA double stranded binding dye: Dyes such as SYBR green are the easiest and cheapest way to monitor PCR in real time and these dyes non-selectively bind to the double stranded (ds) DNA molecule and result in fluorescence.  This fluorescence increases in direct proportion to the amount of PCR product generated in a reaction and be estimated using a sigmoid curve.

B) A fluorescent reporter probe: Generally, this method utilizes the RNA based probe such as Taqman probe with a fluorescent reporter (Green fluorescent protein, GFP) at the 3’ end and a quencher fluorophore at the 5’ end. Once this Taqman probe binds to specific piece of the template DNA after denaturation (high temperature) and the reaction cools, the primers anneal to the DNA. Taqpolymerase then adds nucleotides and removes the Taqman probe from the template DNA. This separates the quencher from the reporter  which ends the activity of quencher and the reporter dye starts to emit fluorescence which increases in each cycle proportional to the rate of probe cleavage. This is then quantified using a computer. The more times the denaturing and annealing takes place, the more opportunities there are for the Taqman probe to bind and, in turn, the more emitted light is detected which is then measured in the real-time PCR thermocycler, This is more widely used method and accurate as the probe is specially designed to be sequence specific and will only bind to the specific PCR product.  

The fluorescence  increases in direct proportion to the amount of PCR product  generated in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template.The light emitted from the dye in the excited state is received by a computer and shown on a graph display, showing PCR cycles on the X-axis and a logarithmic indication of intensity on the Y-axis.

Apart from these two, another method is called ‘Molecular Beacon’ is also used. These are single stranded hairpin shaped oligonucleotide probes that recognize and report the presence of specific nucleic acids in homogeneous solution.  These are  dual-labelled probes which in turn have self-complementary ends that form a stem-loop structure (hairpin) in their native state. The loop contains a probe sequence that is complementary to a target sequence, and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence.

2) Inverse PCR: This variant of PCR, described by Ochman et al., in 1988, enables to amplify DNA with only one known sequence and it overcomes the limitation of normal PCR in which both 5’ and 3’ flanking regions of the desired gene segment must be known. .The method uses the polymerase chain reaction (PCR), but it has the primers oriented in the reverse direction of the usual orientation. . In this methods, the steps involved are: digestion of digestion of source DNA using restriction endonuclease, circularization of double-stranded DNA using self ligation, reopening of the circular DNA by subjecting it to restriction digestion with endonuclease thereby generating a linear product fragment which now will have known region on both it ends and therefore can be amplification using the primers specific to the known region. 
Inverse PCR has numerous applications in molecular biology such as the amplification and identification of sequences flanking transposable elements, and the identification of genomic inserts. 

3) Anchored PCR: This variant is applied to double-stranded DNA fragments for which the sequence at only one end of the gene is known. This method is used in cases where a sequence of only one end of the desired fragment is known for amplification. 

When sequence of only end of the desired segment or gene is known, a primer complementary to the 3’ strand of this end is used to produce multiple copies of only one strand of the gene. In this method, a poly-G or any other photopolymer tail is added to the 3’ end of the ss DNA copies produced by PCR which will serve as template for the daughter strand synthesis. Now to this, a complementary homopolymer, poly-C to be used as a primer for copying the DNA single strands generated by PCR and thereby yields DNA duplex which can then be amplified normally with primers at both the ends

4) Reverse transcription polymerase chain reaction (RT-PCR): This variant can be used to amplify RNA sequences into DNA duplexes. In this method, firstly  cDNA (complimentary DNA) is synthesized from RNA by reverse transcriptase using oligo dT primer which allows the removal of mRNA allowing the second strand of DNA to be formed. 
This reverse transcription RT-PCR-based assays are the most common method for characterising or con???rming gene expression patterns and comparing mRNA levels in different sample populations . RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenza virus A and retroviruses like HIV.RT-PCR is considered to be very efficient, rapid and highly sensitive to virus isolation and is highly recommended as a primary tool for virus detection.

5) Hot Start PCR: In this variant, a critical component like Mg2+ or Taqpolymerase is left out from the reaction mixture and is added usually after the mixture is heated to denatured temperature thereby opening the tops of PCR tubes.The logic behind this method is that most DNA samples used as template will contain some ssDNA and to them primers can pair in an unspecific manner during the preparation of reaction mixture. And as Taq polymerase shows activity at room temperature, it can extend such primers before the temperature is raised for denaturation, resulting in non-specific  amplifications. To avoid these, hot-start PCR was devised.

6) Nested PCR: This variant employs two sets of primers in which the target sequence is amplified using a first pair of primers and then a portion of the amplified product is re-amplified using another pair of primers. The first set of primers  yields a large product, which is then used as a template for the second amplification while the second set of primers anneal  to sequences within the first product thereby yielding smaller second product. The principle involved in the second amplification is that the additional sequences amplified by the first PCR are unlikely to share the sequences for the second pair of primers (nested primers) as well. In such cases, the contaminating sequences will be eliminated from the second PCR product. This variant is highly specific and sensitive as it is unlikely that a non-specific product generated in initial round of PCR would be re-amplified again even in by the second set of primers. Another modification in this variant is Semi-nested PCR, where the basic procedure is same as that of the Nested PCR; however in this case in the second PCR, one of the primers will be a primer which has already used in first round of PCR.. 

7) Multiplex PCR: This variant enables the amplification of multiple DNA targets by addition of more than one set of primers.This methods requires extensive optimization as involvement of multiple primers in a single reaction may increase the risk of primer-dimer formation. The purpose of utilizing this method is that it is possible to amplify multiple segments of the desired DNA fragment at the same time which in turn saves time, minimizes cost and conservation of template DNA is also facilitated. In addition, the annealing temperature of the primers should be similar so that they can anneal and disassociate from the cDNA sequence at the same temperature.  Also, here the amplicons obtained should be of differing length so that can be easily distinguished based on their lengths when run on gel electrophoresis. Moreover, thermo cycling parameters should be optimized which are determined largely by the sequence of the primer sets. Generally, extension times should increase with the number of loci amplified in the reaction. However, very long extensions and annealing times may give rise to non-specific amplifications. [49]. This PCR technique is used for genetic screening, microsatellite analysis, and other applications where it is necessary to amplify several products in a single reaction,  pathogen identification, gender screening, linkage analysis, forensic studies, template quantification and genetic diagnosis of diseases. 

8) Touchdown PCR: This variant of PCR has been designed to increase the specificity of PCR without lowering the efficiency. The PCR is initiated at a very high annealing temperature just below the melting temperature of the primers so that it allows only perfectly matches primer-DNA templates to form and support amplification. The annealing temperature is then subsequently lowered in each cycle of the PCR. The principle involved in this that the first few cycles will amplify only target sequences due to high annealing temperature. These products then will serve as major template for the subsequent cycles; therefore annealing temperature becomes less critical during the later cycles as primers bind less specifically. Thus, higher the annealing temperatures during initial cycles, higher will be the specificity. While lower annealing temperatures in later cycles will increases the efficiency without lowering the specificity.  Any difference in Tm between the correct and incorrect annealing will give an advantage of 2-fold per cycle. This variant is widely used to optimize PCRs, increase specificity and yield by avoiding for lengthy optimizations procedures. 

9) Asymmetric PCR: This PCR is used to generate single-stranded copies of DNA sequence which can then be readily used for DNA sequencing. This is carried out by adjusting the quantities of two primers for the 3’ borders of the target DNA segment in the reaction mixture in such a way that one of the primers gets exhausted at around 10th cycle or so before the termination of PCR.  i.e., it’s used in a very low concentrations e.g., 50:1 ratio. As a result, in the terminal 10th cycle or so, only a single stranded DNA segment will be copied which is considered to be an ideal starting materials for DNA sequencing. This variant is therefore known as Asymmetric PCR. This method is mainly used in certain types of sequencing and hybridization probing where having only one of the two complementary stands is desired.

10) Overlap-Extension PCR: This variant of PCR is used to induce modifications within the internal sites of a gene. In this method, the primers are prepared using the sequence of the site into which mutation is to be induced; base sequence of the primers is altered to induce mutations. Also, these primers are prepared specific to the two 3’end sequences of the gene. The PCR is then carried out in two steps:

a) In first reaction, one 3’ end primer and one internal site primer is used for PCR. b) While in second reaction, the second 3’ end primer and primer of internal site is used for PCR. In both these reactions, PCR will yield one complete and one incomplete strand of the gene. The incomplete strands from the two reactions have a complementary region represented by internal primers. These two incomplete strands are mixed together and are annealed. After annealing of the primers to the template, the synthesis step of PCR extends the 3’ end of the incomplete strands to yield a complete DNA duplex copies containing the specific mutation in the desired internal site of a gene. .

Detection and analysis of the PCR reaction product: 

The PCR product should be a fragment or fragments of DNA of defined length. The simplest way to check for the presence of these fragments is to load a sample taken from the reaction product, along with appropriate molecular-weight markers, onto an agarose gel which contains 0.8-4.0% ethidium bromide. DNA bands on the gel can then be visualized under ultraviolet trans-illumination. By comparing product bands with bands from the known molecular-weight markers, one should be able to identify any product fragments which are of the appropriate molecular weight.

Advantages of PCR: PCR method has many advantages which makes it a desirable tool in molecular biology; some of these advantages are described briefly below: 

1) In PCR, there is very little use of radioactive material during the preparative procedures which makes it relatively safe procedure. However, ethidium bromide and UV radiation are used for which appropriate precautions must be taken.

2) PCR is easy to perform and does not require any specific skill. 

3) PCR is very rapid. It requires only few hours as compared to gene cloning which requires days or weeks.

4) PCR is a highly sensitive technique; it allows the detection of even a single copy of the target sequence present in the DNA samples and thereby generates millions of copies of this sequence. 

5) PCR requires only nanogram quantities of DNA compared to the microgram required by gene cloning. 

6) PCR doesn’t require storage of costly materials such as DNA ligase and Vector DNA as in case of gene cloning; this reduces the cost of PCR.

Limitations of PCR:

Certain limitations of PCR are: In spite of having several advantages, PCR methods faces some limitation such as:

1) Sequence information: The sequence of flanking regions if the DNA fragment to be amplified needs to be known in order to develop PCR primers. 

2) Amplicon size: The DNA fragment amplified by PCR is called amplicons. Generally PCR methods yields amplicons in 0.1–5 kb size range. Although small segments of DNA can usually be amplified easily by PCR, the efficiency of PCR decreases with an increase in amplicon size and consistent results become more and more difficult to obtain.

3) Error rate during amplification: Taq polymerase, a commonly used enzyme in PCR lacks proof reading activity as a result it is unable to rectify the errors committed during the amplification. Generally, all DNA polymerases commit errors during DNA replication, however, they correct themselves by proof-reading i.e. based on their 3’to 5’ exonuclease activity. And these uncorrected errors create problem when PCR products are subjected to cloning. In normal PCR products when are used directly for sequencing, the correct sequence of the amplicon will be attained in spite of high error rate as these errors are distributed randomly; very molecule which has an error at a particular nucleotide position, there will be many molecules having the correct base at that position due to which error will not lead to confusion. However, in cloning, any recombinant clone will have a single random molecule from among the millions obtained by PCR and whether it contains errors cannot be ascertained. So the results obtained from experiments carried out with cloned PCR products are not completely reliable.

Applications of PCR:

PCR has many exciting and varied application in numerous fields such as genetics, forensics, microbiology, clinical diagnosis, etc. Some of these applications are outline below:

1) PCR can be used to amplify a specific gene present in different individuals of a species, somatic cells or gametes like human sperms.  These copies can be used for cloning.

2) PCR is used to study a variety of chronic virus infections (HIV, HCV, hepatitis B virus, human papillomavirus, cytomegalovirus). PCR has been crucial for the detection of HIV infection in neonates, Quantification of HIV and HCV viremia by PCR has been used to monitor response to drug therapies. 

3) PCR is used to identify bacteria and virus infections. It is not only very sensitive but is also selective enough to distinguish closely related strains. For example, this procedure has been adapted to detect genetically modified crops to help ensure they are utilized only in approved ways.

4) PCR can be used to determine the sex of embryos as in case of murine embryos,bovine embryos, Odontocete cetaceans (Whales) embryos, goat embryos, etc. The sex of in vitro fertilized cattle embryos could be determined using Y- chromosome specific primers before their implantation in the uterus. 

5) The use of quantitative Real-Time RT-PCR to detect viral genomes such as HIV (AIDS or HPV (human papilloma virus in association with cervical cancer) is another diagnostic application of PCR. 

6) PCR also finds its use in taxonomy e.g. in microbial taxonomy: Aspergillus section nigri, trichoderma genus, rhizobial strain, etc.  Also, PCR is widely used in plant taxonomy studies and interspecific hybridization e.g., genus, chloroplast genome analysis.

7) DNA fingerprinting is now almost exclusively based on PCR. e.g., Conifer genomes, DNA fingerprinting in aquatic species ofUtricularia L.section have been successfully carried out. ,Rice (Oryza sativa L), Wheat (Triticum aestivum L.) genotypes.

8) PCR is being used to analyse the preserved tissues of extinct species, including ancient human remains. E.g., Tasmanian tigers , ancient cattle etc. By studying the sequence similarities in desired genes, phylogenetic trees can be mapped out. e.g., Bombax mori, Avipox virus, Inbred strains of rat, etc, phylogenetic analysis has been successfully carried out in silk moths using PCR.  Also, PCR is used in forensic analysis..

9) PCR has been able to carry out prenatal diagnosis of genetic diseases, e.g., sickle cell anaemia (one of the first application of PCR).   in diagnosing haemoglobin variants, diagnosis of Phenyl ketonuria , diagnosis of prenatal thalassemia 

9) RT-PCR is widely used to get information on activity of tumour cells and viruses. E.g.; Prostate tumour cells , tumour cells in the peripheral blood of lung cancer patients., Influenza A virus , Plant DNA and RNA viruses.

Basic Protein Chemistry

Coomassie Blue Stain:  (for gels) 
1) Combine 225 ml Methanol with 225 ml ddH2O. 
2) Add 0.5 grams of Coomassie Blue. 
3) Just before use, add 50 ml acetic acid to 10% final concentration. 
4) Stain for 2 hours. Staining time can be shortened by microwaving; ~50 seconds at medium power. 
5) After staining, pour off the stain into a bottle marked Used Stain.  Stain can be re-used once, but fresh 10% acetic acid must be added).

Coomassie Blue Destain:  (for gels) 
1) Combine 25 ml of Methanol with 437.5 ml ddH2O. 
2) Just before use, add 37.5 ml acetic acid to 7.5% final concentration. 

 

3) Destain, plus sponges (pieces of foam packing) for a couple of hours.  It may be necessary to use more than one change of destain.   Microwaving can be used in this stage also, but the details have not been determined.

 

Coomassie Blue Stain:  (for membranes) 
1) Combine 50 ml of Methanol with 50 ml of ddH2O. 
2) Add 0.1 g of Coomassie Blue. 
3) Stain by pouring a sufficient amount to cover the membrane in a petri dish. 
4) Wash the fluid gentle back and forth over the membrane.  Allow it to be immersed for one to two minutes. 

5) Dispose of stain in a manner similar to that for the gel stain.

Dialysis Tubing Prep 
1) Cut tubing to appropriate lengths and place them in a large beaker. 
2) Add enough 5% NaHCO3 (add 50 grams per liter of ddH2O) to cover the tubing. 
3) Bring to a rolling boil and allow it to boil for 15 minutes.  The tubing will float so place a beaker of the next smaller size on top of the tubing. 
4) Rinse the tubing with an excess of ddH2O. 
5) Soak the tubing in 2 mM EDTA (add 10 ml 0.2 M to 1 liter ddH2O) for one hour. 
6) Rinse again with an excess of ddH2O. 
7) Store in 0.05% Azide (add 0.005 ml per liter of ddH2O).
To Pour Stacking Gel…
0.4mls  Acrylamide                    
0.4mls  10x Stacking Buffer        
40uls  10% SDS    
3.12mls  ddH20    
2uls  Temed    

 

34uls  APS   

Make 15% Mini-gels :
**Important** Due to the fact that Acrylamide is being used, it is extremely important to wear gloves throughout this procedure. 
1) Measure out 5.152-ml ddH2O into a sterile 15-ml-tube.
2) Add 4.58-ml Acrylamide. 
3) Next add 1.1-ml 10x Seperating Buffer.
4) Then add 110-ml 10% SDS. 
5) Continue by adding 56-ml APS. 
6) Finally, add 5.6-ml Temed.(Note: Temed is the polymerizing agent so the following steps must be done fairly quickly.) 
7) Use a disposable pipet, homogenize the solution being careful not to cause any bubbles. 
8) Then use the same pipet to fill the glass leaving about 1 inch at the top. 

 

 

9) Finally, add 0.5 inch of H2O-saturated butanol on top of the gel. Note: Leave the unused portion of the solution in the 15-ml tube. Use this to determine if your gel is ready to use (when the tube is polymerized, so is your gel).  

 

Western Blot Basics

Introduction

Western blotting is an improved methodology used in molecular biology to detect a particular protein in a sample of tissue homogenate or extract. This analytical technique is based on the ability of proteins to bind to specific target antibodies. Primarily, Proteins are separated according to the size by using Gel electrophoresis (SDS PAGE) and then transferred to either nitrocellulose or PVDF membrane.

Procedure

Sample preparation is the first step in Western Blotting.

1. Pellet the cells by low speed centrifugation.

2. Lyse the cells using a suitable cell disruption method for eg. Sonication to release the protein of interest.

3. Spin down the lysate to pellet the proteins. The cell debris goes to the supernatant.

4. The proteins are then solubilized using Buffers containing detergents.

5. Collect the washed proteins in a fresh tube and proceed with analysis.

The second step in Western blotting is protein separation using Gel electrophoresis.
The proteins in the sample are separated according to the size on the gel. The protein of interest is then transferred to a membrane which is either Nitrocellulose or PVDF using an electric field or by applying pressure. Usually the membranes are sticky and binds all proteins non-specifically. 
The binding of protein onto the membrane is based upon hydrophobic interactions as well as charged interactions between the proteins and membrane. The nonspecific binding sites on the membrane are blocked by incubating the membrane in proteinaceous blocking buffer. PVDF membrane needs to be soaked in 100% methanol before use.

The next step is Blocking. The membrane is then blocked, in order to prevent non-specific protein interactions between the antibodies and the membrane. This is usually done by placing the membrane in a solution of BSA or Non-fat milk. Once the membrane is blocked, it is ready to incubate with primary antibody. Incubation is done by diluting the antibody in PBS with some protein (BSA) or 5% skim milk to prevent non-specific binding of the antibody.

The diluted antibody solution and the membrane are incubated in room temperature for 1 hour with gentle agitation. The primary antibody recognizes only the protein of interest and binds to it. The membrane is then rinsed with PBS buffer to remove the unbound primary antibody. Then the secondary antibody is incubated with the membrane. It binds to the primary antibody. This secondary antibody is usually linked to an enzyme that can allow for visual identification of where it has bound on the membrane. The membrane is washed to remove unbound secondary antibodies. Since the primary antibody only recognizes the protein of interest, and the secondary antibody only recognizes the primary antibody, if there is stain present on the membrane, then the protein of interest must also be present on the membrane.

Thus, the protein bands on the membrane that are stained contain the protein that is to be detected. Size estimations can be done by comparing the stained bands to that of a pre-stained protein size marker. If a radioactive label is used, the radioactive membrane can be placed against a medical X-ray film. Bands corresponding to the protein of interest will appear as dark regions on the film.

Elisa

ELISA : The enzyme-linked immunosorbent assay (ELISA) is an important tool in for detecting the presence of an antibody or an antigen in a sample. The ELISA's key features are its high level of sensitivity and robustness. It allows easy visualization of results and can be completed without using any radioactive materials because of which it remains a desired assay of choice for routine scientific,medical and veterinary diagnostic assays.

Types of ELISA : There are two basic types known of ELISA:

1) Indirect ELISA : This method is used to determine the presence of a specific antibody in a given specimen. The steps of the general, indirect, ELISA for determining serum antibody concentrations are:   1)The sample containing a known antigen of defined concentration is added to the well of a microtiter plate where it adsorbs to the wells of the plate through charge interactions.  2) A concentrated solution of non-interacting proteins, such as Bovine Serum Albumin (BSA) or casein, is added to the plate wells to block the non-specific adsorption .  3) Test-antiserum is added to these walls and allowed to incubate; if the antibodies in test antiserum are homologous, they bind with adsorbed antigens forming antigen-antibody complex. The wells are again washed to remove any free antibodies. 4) Enzyme-conjugated (labelled) antibodies are now added which link to the antigen-antibody-complex formed in the previous step.  5) The plate is washed again to remove excess unbound enzyme-antibody conjugates.  6) A chromogenic substrate is applied which converts the enzyme to elicit a signal. 8) The result is viewed/quantified using a spectrophotometer.

2) Direct ELISA : This method employs monoclonal antibodies for detection of a particular antigen in a sample.In this approach, the steps involved are as follows : 1) An unlabeled antibody specific to the antigen of interest is immobilized in microtiter well. 2) The antigen is added and allowed to react with the immobilized antibody to form Ag-Ab complex. 3) An Enzyme conjugate is added (that is specific to the antigen) which forms  a double antibody sandwhich 4) The unreacted antibodies are washed away. Thereby, enzyme subsrate is added to elicit a chromogenic or fluorogenic signal.

5) Results are quantified using a spectrophotometer, spectrofluorometer, or other optical device. This procedure is also referred to as, Double Antibody Sandwitch (DAS) ELISA.

Advantages and disadvantages of both Direct and Indirect ELISA :

A) Advantages of using direct ELISA : It is relatively quick as only one antibody is used. Hence, fewer steps are involved. Also, cross-reactivity of secondary antibody is eliminated.

B) Disadvantages of using direct ELISA : By labeling the primary antibody with enzymes, its Immunoreactivity might be affected. Also the labeling for every specific ELISA is time-consuming and a costly affair. Moreover, this method gives minimum signal amplification compared to indirect method.

C) Advantages of using indirect ELISA : Plenty of commercially available labelled secondary antibodies are available. This method generates a stronger signal amplification. Also, many different visualization markers can be used with the same primary antibody.

D) Disadvantages of using indirect ELISA: There are chances of cross-reactivity with the secondary antibody which may give non-specific signals. Also, steps are increased in this method.  Few other variants of ELISA are also available and these are also often used. These are :   

Direct Antigen coating (DAC) ELISA :

1) The antigen of interest is immobilized on the surface of a well. Now, antibody specific to the antigen is added and allowed to react with the adsorbed antigen. Unreacted molecules of the antibody are washed away leaving only the Ag-Ab complex. This constitutes the primary reaction. 2) In the secondary reaction, an anti-immunoglobulin (anti-Ig; an antibody that reacts with all the antibodies belonging to the same class) is added into the same vessel and allowed to react with the Ag-Ab complex which has been previously formed.  3) Now the anti-Ig will bind to the antibody component of the Ag-Ab complex. This anti-Ig is conjugated to an enzyme in such a way that its activity is not impaired. The enzymes usually used in this case is alkaline phosphatase.  4) The unreacted anti-Ig is washed away and finally the substrate of the enzyme is added along with the necessary reagents to develop color due to the enzyme activity. The intensity of the color developed is proportional to the enzyme concentration. Therefore, color intensity is used to determine the amount of antigen or antibody present. This method of ELISA is often referred to as Direct antigen coating (DAC) ELISA.  This method is chiefly useful in estimating the amount of specific antibody such as HIV, present in the antiserum used for the primary reaction. This is the simplest procedure and can be completed within three procedure and can be completed within three hours.   

Sandwich ELISA : This method measures the amount of antigen present between two layers of antibodies, hence called sandwich ELISA. The antigens to be measured must contain at least two antigenic sites that they can bind to the antibody, and at least two antibodies act in the sandwich. This type of ELISA is chiefly used in quantitation of proteins and polysaccharides. The steps involved in sandwich ELISA are described below : 1) Two antibodies that can bind to two different epitopes on the same antigen are required.  2) One antibody is first immobilized on a microtitre well and is referred to as capture antibody. The other antibody is labelled with a suitable enzyme [eg. (HRP), alkaline phosphatase (ALP) etc.] and is referred to as labelled antibody.  3) Nonspecific binding sites are then blocked on the surface. 3) Then a sample (standard and test) containing the antigen is allowed to react with the immobilized antibody in the well. 4) After that the well is washed, and again enzyme linked antibody is added and allowed to react with the bound antigen.  5) Unreacted labelled antibody is now washed out and the enzyme bound to solid support is estimated by adding a chromogenic substrate.  6) The color developed is measured spectrophotometrically, which is directly proportional to the antigen concentration.

Competitive ELISA :

1. Unlabeled antibody is incubated in the presence of its specific antigen.  2. These bound antibody/antigen complexes are then added to an antigen coated well. 3. The plate is washed, so that unbound antibody is removed. The more antigen is present in the sample, the less the antibody will be able to bind with the antigen in the well, hence it leads to Competition 4. The secondary antibody specific to the primary antibody is added. Then this secondary antibody is coupled to the enzyme. 5. A substrate is added and then the remaining enzymes elicit a chromogenic signal. For competitive ELISA, the higher the original antigen concentration, weaker will be the eventual signal .

Reverse ELISA : This variant of ELISA has been recently developed for investigating the West Nile virus envelope protein and how it is able to detect virus-specific antibodies. In this method an antibody is sandwiched by antigens. The advantage of Reverse ELISA over other ELISA techniques is that it has : (i) higher specificity and sensitivity and (ii) the ability to comparably titrate antibodies from different species.  This newer technique uses a solid phase made up of an immunosorbent polystyrene rod with ogives. This entire device is immersed in a test tube containing the collected sample followed by incubation in the presence of a target followed by washing. A second incubation step is then carried out in the presence of a secondary conjugated antibody followed by second cycle of washing and then subsequent color development by addition of chromogenic substrate. Based on the platform of ELISA, this method uses an antibody microarray to capture native antigens. Autoantibody reactivity is then evaluated by differentially labeling patient IgG and incubating these antibodies with the native antigens that are immobilized on the antibody microarray.The essential steps involved in this procedure are discussed below:   1. Preparation: Purify and separate autoantibodies (IgG) from serum. Label them then with either radioactive tagging or a fluorescent dye. Ogives should be carefully coated with solution and an then a substrate indicator should be added later on to measure intensity of monoclonal antibodies present.  2. Extraction: Now, label autoantibodies with native protein on array. Native antigens bound to antibodies on array are extracted from microarray with unique monoclonal antibodies. Array should be incubated with native protein extracts such as cells, tissue, or body fluid, etc.  3. Analysis : Based on the intensity of the dye that appears in assay, we can analyze the concentration. When the process is complete, the assay is then evaluated by its intensity of color which is determined for each well. The amount of color produced co-relates to the amount of primary antibody bound to the proteins on the bottom of the microwells.

Advantages of ELISA:

1) ELISA tests are relatively accurate tests, is easy to perform and gives fast results. 2) It is considered highly sensitive and specific assay compared to radioimmuno assay (RIA) tests.  3) It doesnt involve the use of hazardous radioisotopes. 4) It doesnt need costly instrumentation.  5) Antigens of very low or unknown concentration can be detected since capture antibody only grabs specific antigen.    

Disadvantages of ELISA :  1) One of the major disadvantage is that ELISA is capable of detecting only one antigen at a time, which becomes a limiting factor. 2) Results are dependent on the skills of the technician and the quality of the ELISA kit. 3) Enzyme/substrate reaction is short term so microwells must be read as soon as possible 4) Negative controls may indicate positive results if blocking solution is ineffective as secondary antibody or antigen (unknown sample) can bind to open sites in well.  5) It being an enzymatic reaction, even small quantities of non-specific binding of primary or secondary antibody might result in false signal.   6) As only monoclonal antibodies can be used as matched pairs which recognize one specific binding site i.e. epitope, this procedure can be expensive as monoclonal bodies are costlier than polyclonal bodies and are also difficult to find.

Applications of ELISA : The ELISA procedure has a broad range of applications from disease diagnosis like infectious diseases, autoimmune disease, certain cancers and Alzheimers. ELISA is also useful in detecting potentially hazardous allergens present in food. ELISA can also facilitate drug discovery by identifying drug targets, evaluating disease progression and allows rapid presumptive screening for certain classes of drugs.

1) ELISA has been used successfully in screening and diagnosis of Chagas and human hydatid disease. 2) ELISA has also been able to determine Ig-E based food allergies.  4) Brucella abortus- a highly contagious zoonosis caused by ingestion of unsterilized milk or meat from infected animals or close contact with their secretions, has been diagnosed effectively using ELISA.  5) ELISA has been also used in diagnosis of rotavirus.  6) ELISA has been used as a rapid assay in comparison to other assays in diagnosis of HIV.

7) ELISA has been used in detecting anti-malarial drug sensitivity affectively in case ofPlasmodium falciparum aldolase.

8) Autoimmune disorders have also been effectively diagnosed using ELISA. E.g. antibodies to F-actin (a protein forming the thin filaments in muscle fibres) have been proposed to increase specificity in the diagnosis of autoimmune hepatitis using ELISA.  9) ELISA enables the measurement of LDL receptors successfully.

10) Disorders related with celiac disease have also been successfully diagnosed using ELISA.