Genotyping With PCR
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The document discusses the application of Polymerase Chain Reaction (PCR) in identifying genotypes, outlining its history, principle, and significance in molecular biology. It details the process of genotyping through PCR, including steps like DNA isolation, primer design, and analysis methods such as gel electrophoresis. Additionally, it highlights a specific study on distinguishing Lactococcus lactis subspecies using PCR and its implications for cheese-making.
Genotyping With PCR
- 1. PCR In Identifying GenotypesPresented to - Dr. Hasan Mahmud Reza Course: Biotechnology Course code: PHR 325 Section : 03 North South University Department of Pharmaceutical Science
- 2. Group Members • Sarah Bin Sultana • Jobeda Khanom Lina • Shamim Hossen • Md. Naimul Islam • Stanley Lawrence Palma
- 3. History of PCR (Polymerase chain reaction) •The polymerase chain reaction (PCR) was originally developed in 1983 by the American biochemist Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his pioneering work. •PCR is used in molecular biology to make many copies of (amplify) small sections of DNA or a gene.
- 4. What is PCR? •PCR is a common tool used in medical and biological research labs. It is used in the early stages of processing DNA for sequencing, for detecting the presence or absence of a gene to help identify pathogens during infection, and when generating forensic DNA profiles from tiny samples of DNA. •Using PCR it is possible to generate thousands to millions of copies of a particular section of DNA from a very small amount of DNA.
- 5. Principle of PCR •PCR principle: It is a chain reaction: One DNA molecule is used to produce two copies, then four, then eight and so forth. This continuous doubling is accomplished by specific enzymes known as polymerases, that are able to string together nucleotides to form long molecular DNA strands.
- 6. What is Genotype • The genotype of an organism is the chemical composition of its DNA, which gives rise to the phenotype, or observable traits of an organism. A genotype consists of all the nucleic acids present in a DNA molecule that code for a particular trait. The outward appearance, or phenotype, is the result of interactions of proteins being created by the DNA. • Modern DNA analyzing techniques have made it easier to identify which segments of DNA are responsible for various phenotypes.
- 7. Example of Genotype • Although a particular genotype consists of many nucleic acids, scientists typically represent genotypes with single letters, or two letters in the case of sexually reproducing organisms that receive one allele from each parent. • For example, the trait for eye color could be represented with the letter “E”. Varieties, or alleles, of that trait that are dominant will be designated by capital letters. Therefore, “E” will represent brown eyes. Traits that are recessive are written in lower case. The allele for blue eyes is recessive to the allele for brown eyes, so we can call it “e”.
- 8. Example of Genotype • So there could be 3 genotypes for this eye colour 1) Homozygous Dominant “EE” “EE” = 2) Heterozygous Dominant “Ee” “Ee” = 3) Homozygous Recessive “ee” “ee” = Here, E = Brown eye color e = Blue eye color
- 9. Genotypes consist of nucleotide. In PCR process we can detect variation of DNA sequences . In this picture we can see that it has single nucleotide polymorphism(SNP). Here provide sequence-specific primer(SSP) Single stand DNA(ssDNA) If provide sequence-specific primer-1(SSP-1) Primer extension is occurred. Exact match SNP identify: G Genotyping by PCR
- 10. Genotyping by PCR If provide sequence- specific primer-2(SSP-2) Primer extension is not occurred. Mismatch SNP identify : Unknown After completing the PCR for detecting genotypes, Comparer the length of products using gel electrophoresis
- 11. Restriction Fragment Length Polymorphisms (RFLP) Analysis Typical RFLP analysis comprises five major steps 1.DNA isolation and purification 2.PCR amplification and restriction enzyme digestion 3.Separation and detection of the digested products via electrophoresis 4.Analysis of data to generate the fragment profile for each sample 5.Clustering analysis based on the profile of samples from step 4
- 12. Steps of Mouse Genotyping Know What you are Looking For If we have detailed information about the genomic modification, we can identify the gene sequence under investigation. Pick the Correct Technique for your Mouse Genotyping For all further characterizations of offspring from established mouse lines, use PCR, because it’s quick and easy. If we need higher sensitivity and reliability, use a quantitative PCR with a melting curve analysis
- 13. Steps of Mouse Genotyping Design the Right Primers • The most important component of any PCR reaction is a perfect set of primers. Spend the most time and effort at this step. • If our mice came from a trusted source with a set of genotyping primers, then great. we’re all ready to test them in a PCR. If not, design primer sets like you would for a regular PCR reaction. Material for DNA Isolation • There are several ways to obtain DNA for mouse genotyping. The one we use depends on the established practice in your lab and the quantity of DNA required for the assay. • we can collect DNA from ear tissue (0.5 to 2 mm) using an ear punch
- 14. Isolating Genomic DNA for Genotyping Once we have our sample tissue in hand, it is time to isolate genomic DNA from the cells. PCR Standardize your genotyping PCR the same way we would for any PCR. Some points to consider: • Optimize your PCR primers using positive controls • Titrate the amount of genomic DNA to find the optimum template concentration • Always include wild type and mutant control samples to compare bands on gel or curves in a melt curve analysis • Often, the difference in the band sizes of the wild type and mutant bands will be very small – make sure you get good separation in the gel by using a longer run
- 15. Rapid PCR-Based to Determine Genotype of Lactococcus lactis Subspecies • A highly efficient, rapid, and reliable PCR-based method for distinguishing Lactococcus lactis subspecies (L. lactis subsp. lactis and L. lactis subsp. cremoris). • Lactococcal strains are essential to milk fermentation, especially in the cheese-making process, providing optimal conditions for curd formation and for the development of texture and flavor. • Isolates from cheese starters were investigated by this method, and amplified fragments of genetic variants were found to be approximately 40 bp shorter than the typical L. lactis subsp. cremoris fragments.
- 16. Materials and methods • Recently, a novel criterion for distinguishing L. lactis subsp. lactis from L. lactis subsp. Cremoris has been reported is glutamate decarboxylase (GAD) activity, which has been observed in L. lactis subsp. lactis and not in L. lactis subsp. Cremoris. • The gadB gene encoding L. lactis subsp. cremoris GAD was apparently inactivated by a frameshift mutation resulting from an adenine deletion or a thymine insertion and encoded a nonfunctional protein. • The gadB fragments were amplified by PCR, using the totalL. lactis DNA or the first-strand cDNA as the template.
- 17. Materials and methods • The PCR primers were designed from the published sequence of L. lactis gadB. The sense primer (gadB21) was located within the gadB gene, and the antisense primer (GAD7) was located downstream from the gene. • Each 50 μl of PCR mixture contained 200 ng of genomic DNA, 20 pmol of each primer, reagent mixture, and Ampli Taq gold DNA polymerase . PCR amplification was conducted with a GeneAmp PCR System 2400 • The PCR conditions were as follows: denaturation at 94°C for 9 min, followed by 45 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 60 s, with an additional extension of 7 min at 72°C after the last cycle.
- 18. Conclusion • The polymerase chain reaction is a very helpful way to replicate DNA ,diagnosis certain diseases and identification of genotype. It is very reliable way to test and analyze the DNA and is also very efficient in the sense that is only take a few hours .
- 19. References: • https://www.the-scientist.com/lab-tools-old/genotyping-with-pcr-46437 • https://biologydictionary.net/genotype/ • https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain- reaction • https://aem.asm.org/content/68/5/2209 • https://aem.asm.org/content/66/5/2235?ijkey=438f2836744a06a982e3d4 11eda9c17b3778bcd2&keytype2=tf_ipsecsha • https://www.youtube.com/watch?v=urmkEf5iL6I&feature=share&fbclid=Iw AR0MuB3flXRHZcSHW6YTJda2yB7D0xvFJf0AANLlVZl5nFgYY2D1bP2N05Y