Lecture 2: PCR Techniques for Biopharmaceuticals PDF

Lecture 2 Techniques for in vitro evaluation of biopharmaceuticals Polymerase Chain Reaction (PCR) Dr. Basma Nagy Abd El-Hamid  What is Polymerase Chain Reaction (PCR) forming of new bonds h...

Lecture 2 Techniques for in vitro evaluation of biopharmaceuticals Polymerase Chain Reaction (PCR) Dr. Basma Nagy Abd El-Hamid  What is Polymerase Chain Reaction (PCR) forming of new bonds h bonds Enzyme making copies Type of chemical reaction of any genetic material that progresses in an (DNA) exponential pattern A technique used to amplify, or make many copies of, a specific target region of DNA in vitro (in a test tube rather than an organism).  This target region can be a gene to understand its function, or a genetic marker used by forensic scientists to match crime scene DNA with suspects. The power of PCR is based on the fact that the amount of matrix DNA is not, in theory, a limiting factor. PCR is therefore a technique of purification or cloning.  The goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments. PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology.  The polymerase chain reaction is carried out in a reaction mixture which comprises: 1. The DNA extract (template DNA). 2. Taq polymerase. 3. The primers. 4. The four deoxyribonucleoside triphosphates (dNTPs) in excess in a buffer solution.  Taq polymerase Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase, purified or cloned from a type of bacteria, Thermus aquaticus, which lives in hot springs and resists temperatures above 100°C. at 72 to be active  Its DNA polymerase is very heat-stable and is most active around 70 °C (a temperature at which a human or E. coli DNA polymerase would be nonfunctional). This heat-stability makes Taq polymerase ideal for PCR as high temperature is used repeatedly in PCR to denature the template DNA, or separate its strands.  PCR primers Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis. In a PCR reaction, the experimenter determines the region of DNA that will be copied, or amplified, by the primers she or he chooses.  PCR primers are short pieces of single-stranded DNA, usually around 10-30 nucleotides in length in order to guarantee a sufficiently specific hybridization on the sequences of interest of the matrix DNA. less than 10 is nonspecific higher number is more specific higher than 30 won't react bec it won't find complement one  Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied. The primers bind to the template by complementary base pairing.  Summary Taq polymerase: responsible for propagation of reaction but not start it. Primers: provide starting point for reaction and help chooses the exact portion to be amplified. DNA tempelates. Nucleotides: are the building blocks required for DNA synthesis.  The steps of PCR It is broken down into three phases: 1. Denaturation phase. 2. Hybridization phase with primers. 3. Elongation phase. The products of each synthesis step serve as a template for the following steps, thus exponential amplification is achieved.  cond is imp 1. Denaturation high temp than 80c as above 80 hydrogen bonds breaks down 94°C 2. Annealing ------------ 55°C lower temp than 80 to form stable at 55 hydrogen bond Primers small seq of dna 3. Elongation faster in 2 hr build billions of dna DNA polymerase at 72 °C  The basic steps are: 1. Denaturation Heat the reaction strongly to separate, or denature, the DNA strands. This provides single- stranded template for the next step. It is carried out at a temperature of 94°C, called the denaturation temperature.  At this temperature, the matrix DNA is denatured. The hydrogen bonds cannot be maintained at a temperature higher than 80°C and the double-stranded DNA is denatured into single-stranded DNA. 2. Annealing or hybridization Cool the reaction to a temperature generally around 55 °C so the primers can bind to their complementary sequences on the single-stranded template DNA. Decreasing the temperature allows the hydrogen bonds to reform and thus the complementary strands to hybridize.  The primers hybridize more easily than long strand matrix DNA. The higher the hybridization temperature, the more selective the hybridization, the more specific it is. 3. Extension or elongation Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA. At 72°C, Taq polymerase binds to primed single- stranded DNAs and catalyzes replication using the deoxyribonucleoside triphosphates present in the reaction mixture. The regions of the template DNA downstream of the primers are thus selectively synthesized.  In the next cycle, the fragments synthesized in the previous cycle are in turn matrix and after a few cycles, the predominant species corresponds to the DNA sequence between the regions where the primers hybridize It takes 20 – 40 cycles to synthesize an analyzable amount of DNA (about 0.1 μg).  Each cycle theoretically doubles the amount of DNA present in the previous cycle. PCR makes it possible to amplify sequences whose size is less than 6 kilobases. The PCR reaction is extremely rapid, it lasts only a few hours (2–3 hours for a PCR of 30 cycles).  This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours, depending on the length of the DNA region being copied. If the reaction is efficient (works well), the target region can go from just one or a few copies to billions.  That’s because it’s not just the original DNA that’s used as a template each time. Instead, the new DNA that’s made in one round can serve as a template in the next round of DNA synthesis. There are many copies of the primers and many molecules of Taq polymerase floating around in the reaction, so the number of DNA molecules can roughly double in each round of cycling. start of dna is 36 strand the net result of pcr reaction is the double 2 to 4 hrs  PCR product detection and analysis The product of a PCR consists of one or more DNA fragments (the sequence or sequences of interest). The detection and analysis of the products can be very quickly carried out by agarose gel electrophoresis (or acrylamide). Depending on the reaction conditions, nonspecific DNA fragments may be amplified to a greater or lesser extent, forming net bands or “smear”  DNA fragments of the same length form a "band" on the gel, which can be seen by eye if the gel is stained with a DNA-binding dye. A DNA band contains many, many copies of the target DNA region, not just one or a few copies. Because DNA is microscopic, lots of copies of it must be present before we can see it by eye. This is a big part of why PCR is an important tool: it produces enough copies of a DNA sequence that we can see or manipulate that region of DNA.  For example, a PCR reaction producing a 400 base pair (bp) fragment would look like this on a gel: standard high thickness, higher conc then more specific qylitative and qyantitative  Applications of PCR PCR is used in many research labs, and it also has practical applications in forensics, genetic testing, and diagnostics. For instance, PCR is used to amplify genes associated with genetic disorders from the DNA of patients (or from fetal DNA, in the case of prenatal testing). PCR can also be used to test for a bacterium or DNA virus in a patient's body: if the pathogen is present, it may be possible to amplify regions of its DNA from a blood or tissue sample. 1. Acellular cloning This is one of the most remarkable applications of PCR. It is possible to isolate or to purify a gene without resorting to traditional methods of molecular cloning which consist of inserting a DNA library in a plasmid vector which is then used to transform a bacterial strain whose clones after selection are screened. It is useless to use a cellular system (bacteria, yeast, and animal or plant cell) to amplify the clone.  The technique is much faster and much less random using PCR. The realization of molecular cloning by PCR depends on two major criteria: the choice of DNA extract (matrix DNA) and primers. It permits, especially in a few hours, the “acellular cloning” of a DNA fragment through an automated system, which usually takes several days with standard techniques of molecular cloning. 2. Forensics and Paternity In forensics, PCR is used for the amplification of polymorphic sites, those regions on DNA that are variable among people. Some polymorphisms result from specific point mutations in the DNA, others from the addition or deletion of repeat units. A number of polymorphic sites have been identified and exploited for human identification.  If the suspect's DNA profile and that of the DNA recovered from the crime scene do not match, then it can be said that the suspect is excluded as a contributor of the sample. If there is a match, then the suspect is included as a possible contributor of DNA evidence, and it then becomes important to determine the significance of the match.  Example Suppose that you are working in a forensics lab. You have just received a DNA sample from a hair left at a crime scene, along with DNA samples from three possible suspects. Your job is to examine a particular genetic marker and see whether any of the three suspects matches the hair DNA for this marker.  The marker comes in two alleles, or versions. One contains a single repeat (brown region below), while the other contains two copies of the repeat. In a PCR reaction with primers that flank the repeat region, the first allele produces a 200 bp DNA fragment, while the second produces a 300 bp DNA fragment: You perform PCR on the four DNA samples and visualize the results by gel electrophoresis, as shown below:  PCR also found application in paternity testing. In fact, the same polymorphic sites used by criminalists to explore the involvement of a suspect in a criminal act are also used by testing laboratories to identify the father of a child whose parentage is in dispute.  3. Genetic Research Rapid amplification of tiny fragments of DNA using PCR enabled several techniques such as southern or northern blot hybridization even when the amount of sample material available was very small. Study of gene expression patterns, where in cells or tissues are analyzed in different stages to check for expression of a specific gene.  PCR also assists techniques like DNA sequencing in which segments of DNA from an area of interest can be easily amplified to study genetic mutations and their consequences. The Human Genome Project used PCR to indicate the presence of a specific genome segment in a particular clone. This enabled mapping of the clones and collating results from several laboratories.  Advanced variants of the PCR technique have been found to be useful in chromosomal analysis techniques that can help in early detection of genetic birth defects in children. PCR augments the traditional method of DNA cloning by amplifying tiny DNA segments for introduction into a vector. By altering the PCR protocol, site-directed or general mutations can be achieved in the DNA fragment of interest.  4. Virology In virology, PCR helped detect and characterize the nucleic acids of viruses, which enabled comprehensive viral characterization and a greater understanding of the virus behavior during infection. This understanding immensely helped clinical treatment and enhanced further research on the viruses.  For instance, PCR is used to detect HIV infection at an early phase even before the antibodies are formed. This is also useful for screening blood samples collected for donation.  The COVID-19 PCR test is a qualitative detection of nucleic acid from SARS-CoV-2 in upper and lower respiratory specimens. Results are for the identification of SARS-CoV-2 RNA. It is generally detectable in respiratory specimens during the acute phase of infection. Positive results are indicative of the presence of COVID-19; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status.  Positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information 5. Mycology and Parasitology PCR technology has also found applications in mycology and parasitology, by enabling early identification of the microorganisms, thus aiding efficient diagnosis and treatment of fungal and parasitic infections. 6. Dentistry The PCR technique has become a standard diagnostic and research tool in the field of dentistry. PCR and other molecular biology techniques enable the diagnosis of infectious microbes that cause maxillofacial infections. This helps in the effective management of conditions such as periodontal disease, caries, oral cancer, and endodontic infections. 7. Diagnosis of genetic diseases The use of PCR in diagnosing genetic disease, whether inherited genetic changes or as a result of a spontaneous genetic mutations, is becoming more common. Diseases can be diagnosed even before birth. Examples include: Genetic counselling – screening the parents for genetic disease before deciding on having children  Preimplantation diagnosis – screening for genetic disease before implantation of an embryo in IVF (in vitro fertilization) Screening for genetic disease before birth using tissue samples from the chorionic villus (the membranes found between the mother and unborn baby); fetal tissue from the amniotic fluid (the fluid around the unborn baby); or the small quantities of fetal DNA (DNA from the unborn baby) found in the mother’s bloodstream  Diagnosing inherited or spontaneous diseases, either as a result of symptoms, or because of family history. 8. Detection and diagnosis of infectious diseases PCR can detect infectious disease before standard serological laboratory tests (tests to detect the presence of antibodies), so allowing treatment to start much earlier. Because of this, PCR is also useful for screening donated blood for infections, and is especially useful for infections that are difficult to culture in the laboratory, such as tuberculosis.  Detection of infection in the environment PCR is used to monitor and track the spread of infectious disease within an animal or human population. PCR can also be used to detect bacterial and viral DNA in the environment, for example looking at pathogens in water supplies. 9. Personalized medicine PCR is used in personalised medicine to select patients for certain treatments, for example in cancer when patients have a genetic change that makes a patient more or less likely to respond to a certain treatment. 10. Other uses PCR is used in archaeology, to identify human or animal remains, including insects trapped in amber, and to track human migration patterns; degraded DNA samples may be able to be reconstructed during the early cycles of PCR. PCR can be used to differentiate between similar organisms such as ticks, or work out relationships between different species.