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PCR Lab PowerPoint Presentation

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Published on : Dec 07, 2020 | Views : 1904 | Downloads : 3

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Opening image . Week 15-16 Labs PCR Lab Pre-lab was to learn about PCR – done? We’ll review PCR, and do an overview of DNA sequencing to prepare for your on-line PCR lab this week (see assignment) We’ll discuss RT-PCR for diagnosis of SARS-CoV-2, just FYI Overview of PCR & Sequencing Lab (Week15) Overview of CRISPR & Gene Therapy lab (Wk 16)
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PCR: Polymerase Chain Reaction 1983 (Nobel Prize 1993) Key Concept: DNA is copied in a test tube (in vitro). Every cycle doubles the amount of DNA in the test tube, resulting in exponential amplification of DNA, billions of times in a few hours. Key steps: -Obtain target DNA (<1 ng is OK) -Add dNTPs, primers, and Taq DNA polymerase in Thermocycler. -1. Denature DNA (~95°C), -2. Cool to anneal primers (~50°C), -3. Extend with Taq polymerase (~72°C). - Repeat each 3-minute cycle. 2n cycles: 22 : 4 copies 23 : 8 copies 210 : 1,024 copies (~1 thousand) 220 : 1,048,576 copies (~1 million) 230 : 1.07 x 109 copies (~1 billion) 240 : 1.09 x 1012 copies (~1 trillion) Uses: Forensics, Prenatal diagnosis, Ancient DNA, DNA fingerprinting.
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What is the procedure following a Covid test (nasal swab or saliva)? RT PCR: Reverse Transcription Polymerase Chain Reaction to diagnose SARS-CoV-2. As an RNA virus, the nucleic acids are first converted to double stranded DNA prior to PCR. Reverse Transcriptase (3’  5’) RNA DNA. DNA polymerase creates a double stranded cDNA copy of RNA d) Primers and probes (tagged with fluorescent dyes) are added that bind to specific regions of SARS-CoV-2 DNA Modified from The Worldwide Test for Covid-19 Thank you to Dr. Joon Lee for suggesting this article.
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d) The primers provide a starting point for the DNA polymerase to copy the DNA. The probe (tagged with a dye) recognizes a sequence between the two primers. As DNA polymerase extends, it degrades the bound probe, which results in an increased fluorescence signal as the dye is released. The fluorescence increases as PCR amplifies the virus DNA. With every cycle of PCR, the SARS-CoV-2 DNA signal is amplified in real time (ie: quantitative PCR). If the fluorescence crosses the threshold, the test is positive for SARS-CoV-2.
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3 5 DNA Sequencing: 1977: Sanger Dideoxy DNA sequencing Key ingredient: Chain-terminating Dideoxy nucleotides (ddNTPs) with no 3’OH Combine template DNA to be sequenced with DNA polymerase, all 4 nucleotides, and a DNA Primer. Add a small amount of fluorescently tagged ddNTPs. As DNA is copied, ddNTPs are randomly taken up at each position in the DNA copy, terminating that strand and generating a ladder of fragments that each differ by one nucleotide. The fragments are separated by size, by capillary gel electrophoresis, with a laser detecting each fluorescent dideoxy nucleotide Next Gen Sequencing (Illumina sequencing) uses fluorescently tagged nucleotides, and runs massively parallel reactions on millions of fragments simultaneously. 3 5
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Lab Week 15: PCR, DNA Sequencing, DNA Barcoding with 16S rRNA gene sequences PCR movie (1.5 min) CSHL DNA Learning Center Interactive Animation Briefly go over Virtual Bacterial Identification Introduction (BLAST & Unknown ID) https://media.hhmi.org/biointeractive/vlabs/bacterial_id/index.html Go over K101_PCR_Lab_Assignment_Reflection_FA20.doc (15 points) You will complete & turn this in. Be sure to use your own words.
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Lab Week 16: CRISPR-Cas9 and Gene Therapy Last Quiz (10 points) is just for fun. Two attempts if needed. Briefly go over: The interactive: CRISPR-Cas9 Mechanism & Applications (HHMI; give it a little time to load) Handout (10 points): Using CRISPR to Identify the Functions of Butterfly Genes [PDF] Reflection (5 points): Genes as medicines, HHMI; Be sure to use your own words throughout.
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More Nobel Prizes 1958: For the amino acid sequence of Insulin 1980: Development of Sanger Dideoxy sequencing For PCR For oligonucleotide site-directed mutagenesis And 1980: Two Nobels!
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