Introduction. Definition of PCR. A basic PCR components and reagents
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1 Introduction Off all techniques In molecular biology, the polymerase chainreaction (PCR), hasbeen byforthemostuseful. Haifa Almubarak and Amani Alghamdi PCR is a process in which a DNA sequence is artificially amplified by repeated cycles of replication and strand separation, generating thousands to millions of copies of a particular DNA sequence. The principle of PCR The PCR technique copies the target DNA by performing repeated cycles each containing the following three main steps: 1- A denaturation or melting step to separate the two strands of DNA, this step requires very high temp 95C for seconds. 2-The Annealing step, allowing the primers to bind to the complementary sequences on the template DNA, this step requiresthetemptobedroppedto50-60 C. 3- The Elongation step, once the primers are bound to the template the synthesis of DNA can start, the temperature should be increased to 70c which is the optimum temperature for the polymerase enzyme. Definition of PCR It is a genetic technique that occurs in vitro which allows the enzymatic synthesis of large quantities (amplification)of a targeted region of DNA. The DNA is synthesized in the same manner as that seen in vivo (in the cells ) using a DNA polymerase (the enzymes that cells use to replicate their DNA). A basic PCR components and reagents DNA template: is the DNA molecules that contains the DNA region (segment) to be amplified, the segment that we are concered with is the target sequence. Two primers: a short segment of DNA that are complementaryto the 3' ends of each of the sense and anti-sense strand of the DNA target, they are needed to get DNA synthesis started. A basic PCR components and reagents 1) DNA template 2) Two primers 3) Taq polymerase 4) Deoxynucleoside Taq polymerase: is the enzyme to manufacture the DNA copies. The PCR involves a couple of high temperature steps so we use a heat resistant DNA polymerase, this is extracted from heat resistant bacteria living in a hot springs at temperature up to 80 C, or another DNA polymerase with a temperature optimum at around 70 C. triphosphates 1) Buffer solution 2) Divalent cations 3) Monovalent cation ١
2 A basic PCR components and reagents Deoxynucleoside triphosphates: the building blocks from which the DNA polymerases synthesizes a new DNA strand. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations, magnesium or manganese ions; generally Mg 2+ is used, but Mn 2+ can be utilized for PCR-mediated DNA mutagenesis, as highermn 2+ concentration increases the error rate during DNA synthesis Monovalent cation potassium ions. PCR machine: Also called thermal cyclers, modern PCR machines rapidly changes temperatures for PCR reactions, allowing PCR to cycle between primer annealing, DNA amplification, and strand melting cycles. PCR Primers All DNA polymerases require short segment of double--stranded nucleic acid to initiate DNA synthesis. During DNA replication cells use short stretches of complementary RNA synthesized by enzymes called primases to initiate polymerization. In the laboratory, short, complementary segments of DNA called primers are used in PCR to initiate DNA synthesis and to designate the specific target region to be amplified. The primers (also called oligonucleotides meaning small number of nucleotides) are easily synthesized in the laboratory and can be designed to be complementary to any known DNA sequence. They can range in size from 10 to 100 nucleotides in length, but typically they range from 15 to 30 bases forpcr. It Is The Amplification Primers That Determine Target Specificity (i.e., Which segment Of The Template DNA will get amplified) in the PCR reaction. PCR Procedure (Denaturation) The mixture is heated to break the hydrogen bonds in the DNA, forming single stranded molecules. PCR Procedure The DNA to be amplified is mixes with Deoxynucleoside triphosphates, a thermal stable DNA polymerase and DNA primers. PCR Procedure (Extension) Tag polymerase then synthesizes the complementary strandof DNA.Using theprimerasthestarting point PCR Procedure (Primer Annealing) Themixtureisthencooled sufficiently to allow the DNA primer to anneal to each end of the segment tobecopied The DNA primers hybridizetotheend of the gene to be amplified and provide a starting point for the tag polymerase ٢
3 Denaturation Denaturation: During the denaturation step, the reaction cocktail (reaction mixture) is exposed to high temperature, usually C. This high temperature will denature the DNA meaning the hydrogen bond between the two complementary strands melt, unraveling the DNA molecule and exposing the nucleotide bases. Thehightemperatureof thedenaturing stephastheadded advantage of denaturing proteins (inactivating them) and disrupting cells so that you don t have to always start with purified DNA as your amplification template. You can often amplify DNA directly from cell lysates or even whole cells. PCR Procedure Each synthesis cycle Is composed of Three steps Denaturation Primer Annealing Extension Extension: The reaction cocktail is now brought to the optimum reaction temperature for Taq polymerase (68 to 72 C). During this step, the Taq will bind to each DNA strand and extend from the priming sites (add nucleotides to synthesize a complementary strand of the targeted DNA). Primer Annealing During the second step of each cycle, the temperature is lowered to an annealing temperature, allowing binding (annealing) of the primers to their complementary targets on the DNA template (one for each DNA strand). Theseare designed to flank the desired target region of your DNA template and serve as the starting points for DNA synthesis by the Taq polymerase. Each pair of primers will have a particular annealing temperature determined by the length of the primers and their G+C content. Using the proper annealing temperature for your primer set is essential for efficient and accurate amplification. PCR Procedure (new cycle) The temperature is raised again to separate the DNA strands and the lowered sufficiently to allow the primers to attach. Tag polymerase now synthesizes another set of new complementary strands ٣
4 PCR Procedure This amount of amplification can be achieved by running the reaction overnight in a thermal cycler, an instrument that automatically raises and lowers the temperature at appropriate time intervals PCR Procedure This process is repeated until enough DNA has been produced to be identified or used for further research. After twenty-one cycles, one molecule of DNA can be amplified to over a million copies Applications of PCR It is used in forensic medicine where amplification of the DNA is very useful especially when only trace amounts of DNA is available as evidence, may also be used in paternity testing, can also be used in the analysis of ancient DNA like the DNA analysis of the remains of Egyptian mummies. Applications of PCR PCR is useful when small initial amounts of DNA are available (e.g., forensics or diagnostic samples) or anytime large quantities of a particular region of the genome are needed (e.g., for DNA sequencing or finger printing). 1- Selective DNA isolation; PCR allows selective amplification of a specific region of the DNA which can be used later for many other investigations such as DNA sequencing,genetic finger printing, investing evolutionary relationships among organisms, PCR Optimization: In practice, PCR can fail for many reasons that is why it is important to ensure successful PCR conditions of the reactions and technique should be optimized: Contamination can cause the amplification of unwanted DNAs, thus there should be spatial separation of PCR set up areas from areas of purification and analysis of the PCR product. Add to that all precautions should be taken to minimize contamination as much as possible. Applications of PCR 2- For quantitification of DNA : this allows to estimate theamountof DNA presentin asamplewhich is used to quantitatively determine the level of gene expression. Real -Time PCR is used then which measures the accumulation of the DNA product after each PCR round. 3-PCR In diagnosis of disease; It permits early diagnosis of malignant diseases or can establish whether the person is at risk or not (by investigated the presence of a certain gene that is associated with a certain type of cancer) ٤
5 General Precautions: There should be a spatial separation of PCR setup areas from areas of purification and analysis of PCR. Use of pipette tips with filters (to minimize contamination). storage of materials used in PCR should be separated from all other reagents and should be added to the reaction mixes in a sterile spatially separated facility. PCR Optimization: The primer designing is very important in improving the product yield and avoiding formation of spurious products. Thechoiceof bufferorpolymeraseenzymecan helpin the amplification of long or otherwise problematic regions of the DNA. General Precautions: Use of PCR grade distilled water which has been heat and UV sterilized. Theuseof nonpowderedgloves What is DNA Profiling? A technique used by scientists to distinguish between individuals of the same species using only samples of their DNA (DNA fingerprinting) ٥
6 Step 2: The DNA is cut into fragments using restriction enzymes. Each restriction enzyme cuts DNA at a specific base sequence. Stage 1: Cells are broken down to release DNA If only a small amount of DNA is available it can be amplified using the polymerase chain reaction (PCR) Stage 3: Fragments are separated on the basis of size using a process called gel electrophoresis. DNA fragments are injected into wells and an electric current is applied along the gel. The sections of DNA that are cut out are called restriction fragments. This yields thousands of restriction fragments of all different sizes because the base sequences being cut may be far apart (long fragment) or close together (short fragment). A radioactive material is added which combines with the DNA fragments to produce a fluorescent image. A photographic copy of the DNA bands is obtained. DNA is negatively charged so it is attracted to the positive end of the gel. The shorter DNA fragments move faster than the longer fragments. DNA is separated on basis of size. ٦
7 Uses of DNA Profiling DNA profiling is used to solve crimes and medical problems Stage 4: The pattern of fragment distribution is then analysed. Biological materials used for DNA profiling Blood Hair Saliva Semen Body tissue cells DNA samples have been obtained from vaginal cells transferred to the outside of a condom during sexual intercourse. Crime Forensic science is the use of scientific knowledge in legal situations. The DNA profile of each individual is highly specific. The chances of two people having exactlythe same DNA profile is 30,000 million to 1 (except for identical twins). Example: A Paternity Test By comparing the DNA profile of a mother and her child it is possible to identify DNA fragments in the child which are absent from the mother and must therefore have been inherited from the biological father. Solving Medical Problems DNA profiles can be used to determine whether a particular person is the parent of a child. A childspaternity (father) and maternity(mother) can be determined. This information can be used in Paternity suits Inheritance cases Immigration cases ٧
8 Is this man the father of the child? Mother Child Man ٨
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