Gene Amplification & PCR Process PDF

GENE AMPLIFICATION P OLYM ER A SE CHAI N R EAC T ION 4/21/2024 ASS.LECTURER: SALMA ZAHER 1 Gene Amplification Gene amplification refers to an increase in the number of copies of a gene in a genome. Cancer cells, for example, sometimes produce multiple copies of a...

GENE AMPLIFICATION P OLYM ER A SE CHAI N R EAC T ION 4/21/2024 ASS.LECTURER: SALMA ZAHER 1 Gene Amplification Gene amplification refers to an increase in the number of copies of a gene in a genome. Cancer cells, for example, sometimes produce multiple copies of a gene(s) in response to signals from other cells or the environment. One well-known example of gene amplification and cancer is amplification of the HER2 gene in a subset of breast cancers. HER2 gene amplification results in the production of excess HER2 protein on the surface of the cancer cell. 4/21/2024 ASS.LECTURER: SALMA ZAHER 2 Polymerase chain reaction (PCR) PCR is a technique used to make numerous copies of a specific segment of DNA quickly and accurately. The polymerase chain reaction enables investigators to obtain the large quantities of DNA that are required for various experiments and procedures in molecular biology, forensic analysis, evolutionary biology, and medical diagnostics. 4/21/2024 ASS.LECTURER: SALMA ZAHER 3 4/21/2024 ASS.LECTURER: SALMA ZAHER 4 Principle The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment of DNA is amplified using primer mediated enzymes. DNA Polymerase synthesizes new strands of DNA complementary to the template DNA. The DNA polymerase can add a nucleotide to the pre-existing 3’-OH group only. Therefore, a primer is required. Thus, more nucleotides are added to the 3’ prime end of the DNA polymerase. 4/21/2024 ASS.LECTURER: SALMA ZAHER 5 Components PCR reaction mixture has to include: 1- DNA template 2- Two PCR primers 3- DNA polymerase 4- Deoxynucleotide triphosphates (dNTPs) 5- Buffer solution 4/21/2024 ASS.LECTURER: SALMA ZAHER 6 4/21/2024 ASS.LECTURER: SALMA ZAHER 7 Components 1. (DNA Template): PCR is a highly sensitive technique and requires only one or two DNA templates for successful amplification. 2. (PCR Primers): The PCR requires the knowledge of DNA sequences that flank the DNA template. Primers are short nucleotide sequences (approximately 15–30 bases) that base pair to a specific portion of the DNA being replicated. 3. (Taq Polymerase): Taq DNA polymerase is the most commonly used enzyme for standard PCR amplification. The function of Taq polymerase is to replicate the target DNA. The DNA polymerases recognize primers as start tags. 4/21/2024 ASS.LECTURER: SALMA ZAHER 8 PCR Primer 4/21/2024 ASS.LECTURER: SALMA ZAHER 9 Taq Polymerase 4/21/2024 ASS.LECTURER: SALMA ZAHER 10 Components 4. (dNTPs): Deoxynucleotide triphosphates (dNTPs) are the building blocks from which the DNA polymerase synthesizes a new DNA strand during successive cycles of PCR amplification. dNTPs consist of four basic nucleotides - dATP, dCTP, dGTP, and dTTP. 5. (Buffer): A buffer of the PCR reaction mixture serves as a chemical environment to maintain an activity and stability of the DNA polymerase. The buffer pH is usually between 8.0 and 9.5 and is often stabilized by Tris-HСl 4/21/2024 ASS.LECTURER: SALMA ZAHER 11 Steps There are three major steps in a PCR, This is done on an automated cycler which are repeated for 25 or 35 cycles. “Thermocycler”, which can heat and cool the tubes with the reaction mixture in a very short time. 4/21/2024 ASS.LECTURER: SALMA ZAHER 12 4/21/2024 ASS.LECTURER: SALMA ZAHER 13  Step 1- Denaturation During the denaturation, the double strand melts open to single stranded DNA. The solution contained in the tube is heated to at least 94°C (201.2°F) using a thermal cycler. The heat breaks the hydrogen bonds of the original DNA sample and separates the DNA into single strands (this is termed denaturation of double-stranded DNA). Normally the denaturation time is 1 min at 94oC: it is possible, for short template sequences, to reduce this to 30 sec or less. Increase in denaturation temperature and decrease in time may also work (as recommend 96oC for 15 sec. 4/21/2024 ASS.LECTURER: SALMA ZAHER 14 4/21/2024 ASS.LECTURER: SALMA ZAHER 15 Step 2 - Annealing The sample mixture is then cooled to between 50 to 60°C (122 to 140°F) allowing the DNA primers and the DNA polymerase enzyme to bind to the individual strands of DNA that were separated by the heat (this is termed annealing of the primers). At this point, the nucleotides (A, T, C, G) from the added mixture solution will pair with the individual separated strands of DNA that resulted from the heating process. 4/21/2024 ASS.LECTURER: SALMA ZAHER 16 4/21/2024 ASS.LECTURER: SALMA ZAHER 17 Step 3 - Extension Once joined together, they form a new complementary strand of DNA (termed extension of the DNA). Thus, a new duplicate double-stranded DNA molecule has been formed from each of the single strands of the original sample molecule. The temperature cycles from 95°C to 50 to 60°C. The cycle is then repeated about 35 to 40 times using the thermal cycler which automatically repeats the heating and cooling cycles of the process. Resulting DNA sequence is doubled each time the heating/cooling cycle is conducted by the cycler. Thus, what started out as a single short segment of DNA from one sample can be amplified to form millions of copies after 35 doubling cycles 4/21/2024 ASS.LECTURER: SALMA ZAHER 18 4/21/2024 ASS.LECTURER: SALMA ZAHER 19 4/21/2024 ASS.LECTURER: SALMA ZAHER 20 Step 4: Validating the Reaction Once your PCR reaction has run, there are two ways of determining success or failure. The first is to simply take some of the final reaction and run it out on an agarose gel with an appropriate molecular weight marker to make sure that the reaction was successful and if the amplified product is the expected size relative to the maker. 4/21/2024 ASS.LECTURER: SALMA ZAHER 21 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. 4/21/2024 ASS.LECTURER: SALMA ZAHER 22 4/21/2024 ASS.LECTURER: SALMA ZAHER 23 Advantages of PCR over other diagnostic tests Owing to its high sensitivity. Specificity and speed. PCR provides a method for obtaining large quantities of specific DNA sequences from small amount of DNA, including degraded DNA samples. The specificity of the PCR reaction is imposed by the use of priming oligonucleotides and theoretically focuses the amplification activities on only a small region of the target DNA 4/21/2024 ASS.LECTURER: SALMA ZAHER 24