Week 4 Nucleic Acid Amplification PDF

NUCLEIC ACID 4 AMPLIFICATION outline At the end of the session, the student must be able to learn: a. Nuclei Acid Amplification b. Target Amplification i. Polymerase Chain Reaction c. Amplification Program d. Elements of PCR Cycle i. Denaturation ii. Annealing iii. Extension/Elongation e. Amplificat...

NUCLEIC ACID 4 AMPLIFICATION outline At the end of the session, the student must be able to learn: a. Nuclei Acid Amplification b. Target Amplification i. Polymerase Chain Reaction c. Amplification Program d. Elements of PCR Cycle i. Denaturation ii. Annealing iii. Extension/Elongation e. Amplification By PCR i. First Four Cycles ii. Completed Amplification Cycle f. Components of PCR i. Short oligonucleotide primers ii. DNA template iii. Nucleotides iv. Polymerase v. Buffers vi. Other Components NUCLEIC ACID AMPLIFICATION Nucleic acid amplification is a technique that makes millions of copies of a specific piece of DNA or RNA. This process allows scientists to study genes and other genetic material in more detail. 3 CATEGORIES 1. TARGET AMPLIFICATION 2. PROBE AMPLIFICATION 3. SIGNAL AMPLIFICATION TARGET AMPLIFICATION involves making many copies of a specific DNA sequence. PCR is the first and protOtypical method for amplifying target nucleic acid. POLYMERASE CHAIN REACTION (PCR) is the first and prototypical method for amplifying target nucleic acid Discovered by Kary Mullis in 1983 (Nobel Prize Awardee) The first successful amplification was a short fragment of the Escherichia coli plasmid, pBR322. The first paper describing a practical application, the amplification of beta-globin and anal- ysis for diagnosis of patients with sickle cell anemia POLYMERASE CHAIN REACTION (PCR) is the first and prototypical method for amplifying target nucleic acid Discovered by Kary Mullis in 1983 (Nobel Prize Awardee) The first successful amplification was a short fragment of the Escherichia coli plasmid, pBR322. The first paper describing a practical application, the amplification of beta-globin and anal- ysis for diagnosis of patients with sickle cell anemia BASIC PROCEDURE OF PCR DNA replication in a cell requires an existing double-stranded DNA as the template to give the order of the nucleotide bases: deoxyribonucleotide bases in the form of deoxynucleotide triphosphates (dNTPs) dATP dGTP dCTP dTTP COMPONENTS OF PCR PRIMER / oligodeoxynucleotides Directs DNA synthesis to the desired region dATP, dCTP, dGTP, dTTP Building blocks that extend the primers 50 mM KCI Monovalent cation (salt), for optimal hybridization of primers to template 10 mM Tris, pH 8.4 Buffer to maintain optimal pH for the enzyme reaction 1.5 mM MgCl2 Divalent cation, required by the enzyme 2.5 units polymerase The polymerase enzyme that extends the primers (adds dNTPs) 10^2–10^5 copies of template Sample DNA that is being tested 1. denaturation double-stranded DNA is denatured into two single strands accomplished by heating the sample at 94°C to 96°C for several seconds to several minutes, depending on the template 2. annealing the second step of the PCR cycle where the two oligonucleotide primers that will prime the synthesis of DNA anneal (hybridize) to complementary sequences on the template the primers determine the specificity of the amplification Annealing temperatures will range from 50°C to 70°C and are usually established empirically. 3. extension This is where DNA synthesis occurs. the polymerase synthesizes a copy of the template DNA by adding nucleotides to the hybridized primers. DNA polymerase replicates the template DNA by simultaneously extending the primers on both strands of the template. This step occurs at the optimal temperature of the enzyme, 68°C to 72°C. SUMMARY OF THE STEPS IN PCR denaturation annealing extension temperature 90-96°C 50-65°C 68-75°C (72) time 20-60 sec 20-90 sec 10-60 sec Separation of two DNA strands Binding of the primers of the template Elongation of segment through the action of DNA polymerase purpose components of pcr primer the critical component of the reaction because they determine the specificity of the PCR. Primers are analogous to the probes in blotting and hybridization procedures. single-stranded DNA fragments, usually 20 to 30 bases in length. types of primer FORWARD PRIMER It runs in the 5' to 3' direction t attaches to the bottom DNA strand which runs from the 3' to 5' direction, therefore they are anti-parallel Its sequence is identical to the sequence of the other DNA strand REVERSE PRIMER It runs in the 3' to 5' direction It attaches to the DNA strand which runs from the 5' to 3' direction Its sequence is identical to the sequence of the other DNA strand mispriming aka aberrant primer binding A fragment synthesized as a result of mispriming will carry the primer sequence and become a template for subsequent cycles of amplification consequences: misprimed products will take components away from the intended reaction interfere with proper interpretation of results primer dimers An artifact often observed in the PCR PCR products that are approximately double the size of the primers. consequences: misprimed products will take components away from the intended reaction interfere with proper interpretation of results DNA Template may be derived from the patient’s genomic or mitochondrial DNA or from viruses, bacteria, fungi, or parasites that might be infecting the patient Genomic dna requires 100 ng to 1 ug of DNA for routine clinical analysis Characteristics of best dna template free of contaminating proteins without nicks or breaks Templates with high GC content and secondary structure may prove more difficult to optimize for amplification. Deoxyribonucleotide triphosphate specific nucleotide bases used in PCR They are made up of a deoxyribose sugar, a nitrogenous base and three phosphate groups They serve as the BUILDING BLOCKS for the newly synthesized DNA strands Deoxyadenosine triphosphate (dATP) Deoxythymidine triphosphate (dTTP) Deoxyguanosine triphosphate (dGTP) Deoxycytidine triphosphate (dCTP) A single solution containing a mixture of all four nucleotides is most convenient and lowers pipetting errors compared with four separate nucleotide solutions. storing dNTPs in lower concentrations results in hydrolysis to dNDP and dNMP. These molecules will inhibit the PCR reaction. DNA Polymerase It catalyzes the synthesis of new DNA strands using the primers & dNTPs It adds the dNTPs to the 3’ end of the new strand The same enzyme would also join the nucleotides together to create one continuous strand thermus aquaticus The most common enzyme used in PCR is the Taq DNA POLYMERASE a bacterium that lives in hot springs, and thus, can survive higher temperatures Its maximal activity is achieved around 72- 80°C reaction chambes It is solution with optimal pH and ionic strength 500mM KCl facilitates primer handling 100mM Tris-HCl maintain pH throughout the reaction 15mM MgCl2 magnesium in the form of MgCl2 may sometimes be already part of the reaction buffer magnesium It may be incorporated into the buffer in the form of MgCl2 It is a divalent cation which serves as a CO-FACTOR for DNA polymerase master mix it is a ready-to-use mixture that contains optimal concentration of dNTPs, MgCl2, buffer and DNA polymerase Preparation of components requires PROPER PIPETTING TECHNIQUE to ensure that the exact amount of each component is added and to prevent any form of contamination while transferring them PCR components are added and mixed in a PCR tube. The PCR tube is then placed in a pre- programmed thermocycler. thermocycler It is an apparatus that holds the temperature to be amplified in a heating block (with holes where PCR tubes are placed) other factors to consider Components to be used must be of optimal quality and quantity to ensure that the products to be generated are also of optimal quality and quantity dna template quality It should be FREE OF CONTAMINANTS since these may inhibit amplification QUANTITY The OPTIMAL AMOUNT must be used in the reaction too high: nonspecific amplification too little: little or no product would be produced pcr primer PRIMER SPECIFICITY It should only bind or anneal to the target DNA segment PRIMER ANNEALING TEMPERATURE It should be 5°C lower than the melting temperature of the primer too high = insufficient primer and template binding may occur too low = nonspecific products may be produced more base pair mismatches occur primer length It should be 18-22bp long to ensure primer specificity Length is expressed as the number of base pairs (bp) present gc content The optimal GC content of a primer should be 40-60% The presence of more GC base pairs contributes to the STRENGTH and STABILITY of the primer’s binding to the template This means that more stable annealing occurs if the primers have higher GC contents analysis of amplified products PCR products which are of good quality and quantity may be used in different applications, but before those products are used in other processes, we must first determine if such products were even produced; if the target DNA was successfully amplified by visualizing or analyzing the PCR products purpose: To determine if there is a product To determine if there is only one product, and that there are no other nonspecific products To determine if the product is of the expected size Those products that were amplified however with incorrect sized are not used for further testing 1. agarose gel electrophoresis It is the most widely used method in visualizing PCR products It separates the products based on their size Charged molecules will move through the gel when an electric current is applied They will migrate towards the electrode with an opposite charge Cathode - negative electrode Anode - positive electrode DNA is negatively charged so it will migrate towards the positive electrode (anode) It involves the use of CHEMICAL DYES (such as ethidium bromide) or FLUORESCENT DYES that are added to the PCR mixture in order to visualize the products After electrophoresis, the gel is then placed on an ULTRAVIOLET TRANSILLUMINATOR, where the amplified products will be seen as BANDS on the gel The appropriate size of the products can be determined through the DNA LADDER (AKA: MW size marker) Used as a reference to determine the approximate size of the amplified product 2. dna quantification It is expected that not all reactions are carried out with 100% efficiency, so the number of generated products may not be equal to the initially expected amounts DNA concentration (expressed in ng or ug) may be determined using the following techniques: A. spectrophotometry determine DNA concentration by determining the ABSORBANCE of the PCR mixture b. Gel Electrophoresis FLUORESCENCE INTENSITY of the band is used in estimating the concentration of the products, as they are proportional C. Real-time PCR (qPCR) it can both AMPLIFY and QUANTIFY DNA segments problems encountered in pcr No product formed Problem with reagents Quality of the template DNA is poor Quantity of the template DNA is incorrect Primers are not working Problem with the settings on thermocycler Ex: Incorrect temperature or time Incorrectly sized products possible causes: Sample contamination Sample may have been contaminated with another sample, with other reagent/components, or with other contaminants Primers anneal to an off-target site on the template Primers incorrectly bind to the template Primers bind to other DNA segments and not to the target sequence. It will cause the amplification of the incorrect segment Nonspecific Products Primers anneal to an off-target site on the template Primers bind to other DNA segments and not to the target sequence. Annealing of a primer to itself or to the other primer White & blue are both primers and they are bound to each other instead of being bound to the target DNA template. Yellow binds to itself. False positive Amplification Problematic primer design Problem in the sequence of the primer Sample contamination Amplicons carryover contamination Product from a previous PCR is carried over to the next reaction. This may happen when we use contaminated pipettes or contaminated reagents. Amplicons may contaminate lab equipment/reagents/PPE and these contaminated materials may then introduce those amplicons to the next reaction APPLICATIONS & MODIFICATIONS PATHOGEN IDENTIFICATION It detects the presence of the pathogen at the earliest stages of an infection DNA -> Antigen -> Antibody DNA/RNA is usually the first detectable target to appear after being infected ANTIGENS bacteria or virus specific antigens (ex: proteins) which may be found on the pathogen’s structure. antibodies produced by the infected individual against the pathogen Even if you are infected, if your body has still not produced antibodies against the virus, then a negative antibody test will be obtained. mutation detection There are a lot of diseases which are caused by mutations or genetic variations that can be detected in a specific DNA segment. It aids in assessment of genetic variations based on the presence or absence of an amplicon and/or its length Ex: Amplification of globin genes to detect mutations that may cause hemoglobinopathies (Thalassemia, Sickle cell anemia, etc.,) disease diagnosis Identifying pathogens and detecting mutations may aid in the diagnosis of diseases FORENSIC INVESTIGATION Isolate DNA from samples that are found in a crime scene like hair, blood, or other forensic materials. Trace amounts of DNA from forensic evidence can be amplified to match the sample to a particular individual PCR METHODS All of these employ the same basic principles and procedures. However, modifications were made in order to improve their functions to make them more useful for different applications. REVERSE TRANSCRIPTION-PCR (RT-PCR) mRNA → cDNA → DNA amplification Starting material: RNA Requires “reverse transcriptase” which converts mRNA into complementary DNA (cDNA) It is currently used for the detection of SARS-CoV-2 REAL TIME-PCR (qPCR) It is also called the quantitative PCR Can perform both amplification and quantification using one equipment in a single reaction It measures the amount of amplified material as it is generated at each cycle Fluorescence intensity increased proportionally to the amount of dsDNA generated MULTIPLEX PCR It uses more than 1 primer pair to allow MULTIPLE AMPLIFICATION of multiple targets simultaneously Saves a lot of time and a lot of resources Ex: 1 sample can be used to test for more than 1 type of swab (1 genital swab can be used to test for different STD-causing organisms) 0 1 sample = test for >1 virus