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Thursday, May 30, 2013

12 Things to rememeber while Setting up a PCR Reaction

12 Things to rememeber while Setting up a PCR Reaction
This is a guest post by one of the blog readers.

setting up a PCR reaction
  • Consumables for setting up a PCR reaction such as PCR Tubes, Pipettes, PCR Racks should be cleaned with Hypo and then expose to UV for atleast 20 minutes.
  • Before Setting up a PCR reaction Wash Hands,Wear Gloves.
  • Always use Aerosol barrier tips (ART - Aerosol Resistant pipette Tips) for setting up PCR reaction, changing tips for each step.
  • Use PCR grade water for diluting the stocks and for setting up a PCR Reaction.
  • Use separate rooms for performing Mastermix preparation, Sample addition & Post PCR.
  • Always use fresh aliquot of Reagents.
  • Reagents for PCR should be thawed completely,vortex it and centrifuge before use.
  • Never enter Post PCR room before setting up your Reaction.
  • Accurate Pipette handling is must - Use calibrated pipettes.
  • Start the PCR Reaction immediately after setting up the reaction this will help in avoiding Primer dimer formation.
  • Positive and Negative controls should be kept in each PCR reaction without that PCR reaction is not considered as valid.
  • Always perform PCR in Duplicates or in Triplicates.
  • Make Sure you have cross checked the calculations before proceeding with the reaction.
Contamination in PCR lab is a Disaster....!!! Handle PCR Products (amplicon) Carefully.

Monday, May 27, 2013

Troubleshooting your PCR Reaction

This is a guest post from one of the blog readers.

In a PCR reaction various things can go wrong, So interpretation and troubleshooting a PCR reaction is important in order to further proceed with the PCR results:

qPCR amplification curve, pcr amplification


Possible Issues


1. No Ct Value / Signal:
This is a common problem faced by many researchers in the lab. PCR reaction is setup and at the end of PCR reaction No reult (No Ct / No Signal), waste of reagents and time.


Possible cause:


  • Problem with PCR mastermix.
  • Forgot to add template DNA
  • Template DNA used is degraded.


Solution:


  • Perform PCR reaction in duplicate or Triplicate.
  • Make sure template DNA is added.
  • Keep a Positive Control and Negative control along with Samples.
  • Re-do the reaction with fresh set of reagent stocks.


2. Evaporation:
Modern PCR mchines will not be having this issue, but the older ones do have, evaporation occurs during the temparature cycling in a PCR reaction, as the temperature can reach upto 95oC causing the mastermx to evaporate from the PCR tube.


Solution:


  • Use mineral oil, Silicone oil or Paraffin beads to prevent evaporation..


3. Poor or Low Signal in real time PCR:
Poor signal can be due to the following reasons


  • Degraded Probe.
  • Probe concentration sub-optimal.
  • Tempalte DNA having Salts (carried over from extraction).
  • Higher concentration of Template DNA.


Solution:


  • Use Fresh aliquot of Probe
  • Optimize Probe concentration
  • Re do PCR reaction by diluting template DNA.


4. Amplification in Negative Control:
The negative control used in a PCR reaction is to verify the reaction has any contamination. Negtive control used will have no template DNA, so nothing should amplify. If something does amplify, it is due to contamination in the reagents, and indicates that the bands in the other samples are likely contaminated as well.
The negative control would be water in place of DNA.
The PCR experiment is not valid without a Negative control.


Causes:
  • Negative control is contaminated with template DNA.
  • PCR Buffers and reagents are contaminated.
  • Primer Dimer formation (Rare chance)


Solution:


  • Prepare fresh set of reagents.
  • Clean working areas, Pipettes with hypo.
  • Use gloves while working.
  • Have separate areas for mastermix preparation, Sample addition and Post PCR analysis.


5.Variation in PCR duplicates


Cause:


  • Inaccurate Pipetting


Solution:


  • Pipette calibration.
  • Follow Proper Pipetting technique.


6. Poor PCR Efficiency
Poor PCR efficiency can be due to many factors in a PCR reaction to list a few


Possible Cause


  • Inhibitors
  • Sub-optimal Primer Probe ratio
  • MgCl2 Concentration not optimal
  • dNTP concentration
  • Inacurate Pipetting
  • Taq Concentration
  • Problem with annealing temparature


Solution:


  • Optimize PCR Buffer, Mgcl2, dNTPs, Taq
  • Try with different annealing temperatures.
  • Pipette calibration.


PCR Product analysis on Agarose gel

In End point PCR or conventional PCR running an Agarose gel is the only way to analyze the PCR product.Even in the initial optimization stages of real time PCR, Agarose gel is performed to analyze the PCR amplification. What all can go wrong when you run a PCR product on Agarose gel.

PCR Product smear gel





  • Smeared Band

Due to higher concentration of DNA / Presence of impurities, this can be solved by diluting the sample before loading.


  • No or Faint Band
Due to No amplification or Poor amplification - this can be solved by optimizing PCR buffers and primers for the particular target.


  • Multiple Bands

Due to primer dimer formation - This can be solved by :


  1. Using Hot Start Taq Polymerase
  2. Optimizing MgCl2, KCL concentration
  3. Primer Specificity
  4. Designing new Primer set with optimum GC content.

Tuesday, May 21, 2013

Importance of Column Packing & Efficiency Calculations

Column Packing is one of the most important step in the chromatographic purification process. A well packed column yields higher resolution and yield.

Column Packing Efficiency can be checked by calculating:
  • HETP - Height Equivalent Theoretical Plates
  • Peak Asymmetry Factor (AF / As)
HETP Calculation:

HETP = ( d2 * L ) / (5.54* t2)

Where: t = retention value expressed as units of time, volume or measured distance on the Chromatogram
d = peak width at half height expressed in same units as “t”
L = column length (in units of cm for HETP or m for N/m)

N/m =  (5.54* t2) / ( d2 * L )

Asymmetry Factor (AF / As) Calculation:


chromatography peak asymmetry



AF = b/a

Where: a is the peak width (left half) at 10% of peak height, b is the peak width (right half) at 10% of peak height, 


Column Packing Guidelines - TOSOH Biosciences 




References
TOSOH Biosciences - Technical Resources
GE Lifescience - Technical Resources
Pall Lifesciences - Technical Resources

Monday, May 20, 2013

TaqMan Assay Vs SYBR Green Assay

Before going into the details of the Assays let's have some basics on real time PCR,

Real Time PCR differs from the convention PCR/ End point PCR is that in real time PCR both amplification and quantification of the target DNA molecule is possible.

In real time PCR the amplified DNA is detected in real time, where as in convention PCR detection is possible only at the end of PCR by performing agarose gel electrophoresis for the PCR product.

TaqMan Assay


Taqman real time pcr

TaqMan assay includes an oligonucleotide probe called the TaqMan Probe, containing a fluorescent reporter dye at the 5' end and quencher at the 3' end. This oligonucleotide is developed in such a way that it will bind to downstream of the primer binding site in the target DNA molecule.

TaqMan assay utilizes the nuclease activity of taq DNA polymerase, during the elongation step in the PCR, taq DNA polymerase displaces the bound reporter dye.

Removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process.

when the reporter and quencher are inn close proximity, the flourescence emitted by the reporter dye will be readily quenched by the quencher, when the reporter is cleaved by the taq DNA polymerase, reporter moves away from the quencher thereby no quenching occurs, resulting in the emission of flourescent signal.

When the PCR cycles proceeds, resulting in more amplification of the target molecules proportionally fluorescent signal will also increase.

Advantages of TaqMan Assay
  • Specific hybridization between probe and target is required to generate fluorescent signal
  • Probes can be labeled with different, distinguishable reporter dyes, which allows amplification and detection of two distinct sequences in one reaction tube.
  • Post-PCR processing is eliminated, which reduces assay labor and material costs
Disadvantages of TaqMan Assay

The main disadvantage of TaqMan assay is that synthesis of different probes is required for different sequences sometimes makes it difficult.

SYBR Green Assay

SYBR Green dye, a dye which binds to the double stranded DNA, helps in detection of the amplified product. SYBR Green dye shows increased flourescence when binds to a double stranded DNA

During the PCR, Taq DNA polymerase amplifies the target DNA molecules, when SYBR green is present it binds to the double stranded DNA and gives flourescence siganl, as the PCR proceeds more and more amplification occurs and there by more binding of SYBR Green to the double stranded DNA resulting in the increase in flourescent signal.



Advantages of SYBR Green Assay
  • It can be used to monitor the amplification of any double-stranded DNA sequence.
  • No probe is required, which can reduce assay setup and running costs, assuming that your PCR primers are well designed and your reaction is well characterized.
Disadvantage of SYBR Green Dye

The primary disadvantage is that it may generate false positive signals; i.e., because the SYBR Green dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences. Therefore, it is extremely important to have well-designed primers that do not amplify non-target sequences, and that melt curve analysis be performed.

Taqman Vs SYBR Green - Lab Rap Battle - Video by Life Technologies Corp


References
Life Technology - Technical Resources
TaqMan & SYBR Green are registered Trade Marks of Invirogen (Life Technologies Corporation)

Sunday, May 19, 2013

Top 5 Revolutionary Biotech Innovations


Recombinant DNA Technology:

The advent of Recombinant DNA technology has revolutionized the biotech and healthcare sector. The impact of this technology is un-imaginable.

recombinant DNA technology

The DNA/gene of interest or foreign DNA can be inserted into a Vector / plasmid and can be expressed in suitable host (Bacterial, mammalian or Viral Systems) to generate the product of interest.
One such product is recombinant human insulin. recombinant human Insulin was the first drug to be licensed, and was developed by Genentech and licensed by Eli Lilly Company.
List of few rDNA technology Products:
  • Erythropoietin (EPO)
  • Granulocyte colony-stimulating factor (G-CSF)
  • alpha-glactosidase A
  • alpha-L-iduronidase (rhIDU; laronidase)
  • N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase)
  • Dornase alfa,
  • Tissue plasminogen activator (TPA)
  • Glucocerebrosidase
  • Interferon (IF) Interferon-beta-1a,  Interferon beta-1b
  • Insulin-like growth factor 1 (IGF-1)

Polymerase Chain Reaction:

PCR or Polymerase chain reaction was developed Kary Mullis in 1983. One of the breakthrough developments in the field of Biotechnology. Development of PCR made genetic identification (Disease diagnosis, Forensic Identification, etc.) a lot more easier than before.

PCR

PCR performed using a thermo cycler, involves the repeated cycling of heating and cooling of the reaction DNA denaturation and enzymatic reaction for the amplification of DNA. minute quantities of DNA can be amplified and detected using this powerful technique.
Applications of PCR
  1. Disease Diagnosis
  2. Forensic Science
  3. Amplification & Quantification of DNA

Stem Cells as Regenerative Medicine

Stem cells are undifferentiated cells which can differentiate to perform specialized functions...There are two different types of stem cells embryonic and adult stem cells.

stem cell treatment

Stem cells therapy can be used for treating nuero degenerative diseases like Alzhimer’s disease, Parkinson's diseases, etc.
Stem Cell Therapy has shown to give promising results and there are no other alternatives in some cases, but most of the therapy procedures are in the initial phase of clinical trials and need more study to analyse the results before it can be applied in wide variety of disease conditions.
As there are many controversies involved in it, lot more research need to be done before it can be applied routinely, Stem cell therapy has the potential to revolutionize the way we treat diseases.

Genome Sequencing Technology:

Genome Sequencing technology has helped to unlock the secrets of the genetic code. Nowadays complete genome sequencing is possible. By sequencing the complete genome enormous amount of information can be obtained. with the advancement of bioinformatics tools, analysing the genome sequence and comparing with genome of different species are possible and easy.

genome sequencing

DNA sequencing can be used to find out the sequence of individual genes or group of genes, full chromosomes or the complete genome. Sequencing provide the order of nucleotides in the DNA or RNA of the cell which can be used for molecular biology research or to make disease diagnosis and treatment easy.

Innovative Drugs:
The way we treat diseases is changing day by day, with the advancement of better therapies and vaccines to boost the healthcare sector. One of the top innovation to be pointed out is the Magic Bullet.


MonoclonalAb


Magic Bullet:
Ehrlich is the scientist who developed the Magic Bullet. The compound which only targets the bacteria which will not harm other cells. Nowadays we can see Monoclonal antibodies which is used in the treat many disease which is to some extent is the modified form of Magic Bullet. Monoclonal antibodies which specifically targets certain type of cells or proteins and initiates cellular immune response to fight against the disease.

List of Some of the FDA approved Therapuetical Monoclonal Antibodies

Adalimumab – for Autoimmune disorders

Bevacizumab – for Colorectal Cancer

Daclizumab – for Transplant Rejection

Ipilimumab - for Melanoma

There are many other Biotechnological Innovations, but i feel these are the five innovations that played major role in revolutionizing biotechnology and healthcare sector.

Friday, May 17, 2013

Global Warming: A major Threat on Climate Change - An International Issue

Global Warming: An international issue
Global warming has great impact on our economy, health and community. If we don't act aggressively now the results can be disastrous. 


global warming


Green house effect, the major cause of global warming, is actually the warming up of earth’s surface & atmosphere due to the effect of CO2 and other gases in the atmosphere.Green House is essential for maintaining the balanced temperature and climatic conditions of the earth. When solar radiations falls on the earth’s surface and reflects, a part of the radiations is absorbed by CO2 and other gases and re-emits to earth results in warming.


global warming: green house gases


Now a day more amount of green house gases [CO2 , CH4, N 2O] are released to the atmosphere through burning of fossils fuels, coal mines, deforestation, motor vehicles., etc. Since more CO2 is released more temperature rise will occur & affects the whole climatic condition of the earth. Some region experience low rainfall and other regions experience High rainfall, resulting in drought, flooding etc.Rise in temperature will melt ice of polar region causing rise in sea level. 

Global warming is also threat to polar bears as ice melts earlier polar bear's life will be put into extinction, as the polar bear cant hunt as the ice melts.


global warming - polar bear extinction

The average facade temperature of the globe has augmented more than 1 degree Fahrenheit since 1900 and the speed of warming has been almost three folds the century long average since 1970. This increase in earth's average temperature is called Global warming. The human actions, mainly the discharge of green house gases from smokestacks, vehicles, and burning forests, are perhaps the leading power driving the fashion.are the cause of Global Warming.

How Global Warming is going to Affect Everyone of us????




Video Source: Natural Resources Defense Council

How can we minimize Global Warming??
Yes, we can minimize the effects of global warming if we act now aggressively.


We cannot stop global warming suddenly but by taking necessary actions we can reduce it gradually. Planting more trees, reducing combustion of fossil fuels can reduce the effects of global warming.
Prevention of deforestation & the use of alternate energy source like solar energy, BiogasBiofuels, etc. should have to be utilized for reducing the effects of Global Warming.

Global warming solutions
Some of the possibilities are:
  1. Developing Energy efficient technologies
  2. Use Low-Level Carbon releasing fuels in transportation vehicles.
  3. Use of Nuclear Energy as an alternative source of energy.
  4. Developing Low or Zero carbon emission technologies.
  5. Boosting Forests and Agriculture.
Even though there are few options and possibilities to minimize global warming, All these options will take time to have some impact on the growing global warming rate.

what will you do to minimize the global warming today???


Tuesday, May 14, 2013

50X TAE Buffer Preparation - Tris Acetic EDTA Buffer

TAE Buffer- Tris Acetic EDTA Buffer

TAE Buffer consists of Tris Base, Acetic acid and EDTA, a buffer which is most commonly used as running buffer for agarose gel electrophoresis in the separation of nucleic acids (DNA and RNA).

Preparation of 50X TAE Buffer Stock Solution

Chemical
Weight / Volume
Molarity
Tris Base
242 gms
2 Molar
Acetic Acid
57.1mL

0.5M EDTA pH 8.0
100 mL
50 milli Molar
MilliQ
up to 1000 mL


Final Concentration in 1X TAE Buffer
Tris – 40mM
EDTA - 1mM

Preparation 1X TAE From 50X TAE Stock

This can be calculated using the formula:

C1*V1 = C2*V2

C1 - Initial Concentration
V1 - Initial Volume
C2 - Final Concentration
V2- Final Volume

For Example, you want to make 1X TAE Buffer from 50X TAE Buffer Stock then how much 50X TAE is required,
50 * V1 = 1*300
V1 = 300 / 50 = 6mL
So to make 300mL of 1X TAE Buffer you should take 6mL of 50X TAE Stock solution and make it up to 300mL using MilliQ water (6mL of 50X TAE + 294mL of Water).

Sunday, May 12, 2013

Azocasein Protease Assay

The azocasein protease assay is done for the activity of protease in the culture broth or in other protein samples, if protease is present it will chew up all the proteins.
Azocasein Protease Assay Protocol
  1. 200uL of 2% Azzocasein in sodium bicarbonate buffer pH 8.3
  2. 200uL of 0.5% sodium bicarbonate buffer
  3. 100uL of Sample to be tested
  • Mix all the above and incubate the tube at 37oC for 10mins.
  • Add 0.5mL of 20% TCA to each tube.
  • Vortex and incubate at room temperature for 5 mins.
  • Centrifuge at 10000 RPM for 5mins/4oC.
  • Add 500uL of supernatant to 1mL of 1M NaOH
  • Read the Absorbance at A440 using a spectrophotometer.
  • Check for the protease activity.

New Product Development

New Product Development

New Product Development or NPD is the term used to describe the complete process of bringing a new product or service to the market.

The New Product Development Process Involves

  1. Idea Generation
  2. Idea Screening
  3. Concept Development & Testing
  4. Business Analysis
  5. Beta Testing
  6. Technical Implementation
  7. Commercialization
Product development portfolio

Idea Generation:

This Involves SWOT Analysis, SWOT stands for

S - Strength
W - Weakness
O - Opportunitie
T - Threat

Concept Development &Testing:

In this stage, Marketing and Engineering details needs to be developed.

Wednesday, May 8, 2013

Explore your Protein Purification Skills

Explore your Protein Purification Strategies using this Free Online Application developed by Andrew Booth, Faculty of Biological Sciences, University of Leeds, UK

application for protein purification


This application helps you put your protein purification skills to work, you can choose no. of proteins to be separated and you will have the option of choosing the Protein method from the list:
  • Ammonium Sulfate Precipitation
  • Ion Exchange Chromatography
  • Hydrophobic Interaction Chromatography
  • Gel Filtration Chromatography
  • Affinity Chromatography
Protein Analysis Methods in the application includes:
  • 1-Dimensional PAGE
  • 2-Dimensional PAGE
  • Commassie Blue
  • Immunoblot
Other Techniques includes
  • Dilutions
  • Enzyme activity Assay
  • Pooling of Elute, Etc.
A nice application to apply your skills on the Protein Purification, why wait check it out.
Run the Application...Its Free!!!

Monday, May 6, 2013

Line Probe Assay - Rapid MDRTB Screening Assay

Line probe assays (LPAs) for tuberculosis were endorsed by the WHO in 2008 for molecular detection of drug resistance from smear-positive patients at risk of Multi Drug Resistant-Tuberculosis (MDRTB)
Two commercial LPAs are currently available:
  • INNO-LiPA Rif.TB test (Innogenetics NV, Gent, Belgium) and 
innogenetics line probe assay

GenoType MTBDRplus test (Hain Lifescience GmbH, Nehren, Germany).
line probe assay


Line Probe Assays for tuberculosis uses PCR/hybridization technique to identify members of the Mycobacterium Tuberculosis while simultaneously identifying drug-resistant strains by detecting the most common single nucleotide polymorphorisms (SNPs) associated with resistance. Meta-analyses have shown that LPAs are highly accurate for the detection of first-line drug resistance, especially in smear-positive sputum specimens.
WHO analysis of systematic reviews and meta-analyses showed that Line probe assays for tb are highly sensitive (≥97%) and specific (≥99%) for the detection of RIF resistance (Rifampicin resistance), alone or in combination with Isoniazid also known as isonicotinylhydrazine (INH) (sensitivity ≥90%; specificity ≥99%), on isolates of M. tuberculosis and on smear-positive sputum specimens.
Accuracy for detection of Multi Drug Resistant-TB was 99%, which remained unchanged when RIF resistance alone was used as a proxy marker for MDR-TB. Hain Lifesciences released the GenoType MTBDRsl test in 2009, designed to test for resistance to second-line anti-TB drugs (fluoroquinolones, ethambutol, aminoglycosides and cyclic peptides), and which can be used in combination with the MTBDRplus test to identify XDR-TB.

Advantages of Line Probe Assay for tuberculosis
  • It can be tested directly in smear-positive sputum samples.
  • It gives rapid drug susceptibility results without the need for culture.
  • LPAs can be used as the primary method for Drug Susceptibility Test on cultured isolates of M. tuberculosis, replacing phenotypic DST.
Disadvantages
The major disadvantages of LPAs are
  • It is labour intensive
  • It require highly trained personnel and dedicated laboratory space and equipment.
References

Wednesday, May 1, 2013

Labelling of DNA / RNA

Labelling of DNA & RNA can be done using:

  1. Radioactive isotopes
  2. Non-Radioactive isotopes
commonly used radioactive isotopes are : 125I, 14C 15N, 35S, 32P, etc.
Labelling can be done using 3 methods
  1. Nick Translation
  2. End Labelling
  3. Random Primer Labelling

Nick Translation

  • Rapid, easy and inexpensive method.
  • Amount of DNase  I added is critical
  • Reaction stopped using EDTA.
  • Purification can be done either by Column chromatography or by Gel Extraction.
Nick Translation

End Labelling

  • End labelling can be done by two ways: 3’ End Labelling and 5’ End Labelling.
End Labelling
  • The Selected DNA is digested with restriction enzymes to generate sticky end.
radioaactive end labelling

Disadvantages of Radioactive Isotope Labelling

  • It is hazardous
  • Require long exposure times
  • Costly
  • Instability due to the half life period of radioactive isotopes.
Non Radioactive Isotope DNA Labelling
  • Safe.
  • Stable.
  • Economical.
  • More stable compared to radioactive isotope labelling.
There are two methods of Non radioactive isotope labelling
  1. Labelling with Biotinylated nucleotide
  2. Labelling with HRP conjugate
nonradioactive labelling

Disadvantages of Non Radioactive Isotope Labelling

  • Less Sensitive

Restriction Digestion

Restriction Digestion

This is an explanation for performing Restriction Digestion using restriction digestion enzymes, some of the examples of Restriction enzymes are E.coRI, BamHI, SalI, etc. each of these enzymes can specifically recognize particular regions of the target DNA and cleave that region to give fragments of different lengths. Single and Double restriction digestions are usually performed for molecular biology cloning related works. there are various restriction digestion enzymes are available suitable one need to selected depending on the target sequence and the product required.
Restriction digestion reaction volume will be usually 50uL, containing the target DNA, restriction enzyme and the assay buffer. The reaction mixture is incubated for 1hr at 37oC, an agarose gel electrophoresis is performed after the incubation period to check the restricted fragments. it is recommended to have a control reaction with out any enzyme. while performing agarose gel electrophoresis don’t forget to load molecular weight marker for comparison of molecular weight of  the restricted fragments.
Restriction digestion

PCR & Site Directed Mutagenesis

PCR – Polymerase chain Reaction

Polymerase chain reaction or PCR is a powerful tool for amplifying a stretch of DNA.From a single piece of target DNA one can make enough copies for sequencing, cloning or for gel electrophoresis.
PCR is performed generally in 0.2 or 0.1ml tube in a thermo cycler, a machine which maintains temperature as per the users input. The PCR reaction mixture or the master mix will have the following components:
  • Target DNA; Isolated from either of these samples :infected Blood or sputum, Tissues, Hair, etc.
  • Primers – Specific to the target DNA to be amplified.
  • dNTPs
  • Taq DNA polymerase
  • Some times PCR enhancers like DMSO, BSA, etc.
Polymerase chain reaction has 3 steps:
  1. Initial Denaturation: Temperature is raised to above 90oC, which breaks Hydrogen bonding between the two strands resulting in strand separation
  2. Annealing:In this step temperature is lowered resulting in the binding of primers to its complementary sequence in the target DNA.
  3. Extension: Here in this step temperature is raised to 72oC there by Taq polymerase adds up the nucleotides to the primers in 5’ to 3’ direction.
DNA duplex is produced after this process, this continues for 40 cycles. One copy becomes two, two becomes 4, and so on.
The no. of copies generated after n cycles are given by the general formula 2n.
if there are X copies in the beginning and after n cycles it will be X*2n copies.

Site Directed Mutagenesis

The primer used in the reaction mixture do not have to match exactly they can still anneal with few number of point mutations which the base pairs are not complementary to the target DNA.
Thus the amplified products will have the mismatched sequence, over the no of cycles proceeds there will be more copied of DNA which has the new sequence in it than from the original template DNA.
This is a convenient way of making changes to the DNA. If the new sequence or the altered sequence is coding for protein then the same amplified product can be cloned and expressed.

Bacterial Growth : Log Optical Density Vs Time is Plotted


Bacterial growth rate can be plotted as follows:
Hours after Inoculation
OD 550
Log OD 550
0
0.008
-2.096910013
1
0.02
-1.698970004
2
0.052
-1.283996656
3
0.135
-0.869666232
4
0.2
-0.698970004
5
0.282
-0.549750892
6
0.447
-0.349692477
7
0.661
-0.179798541
8
1.122
0.049992857