l10 Northern Blotting Techniques in Genetics
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Questions and Answers

What is the primary function of SYBR Green I in molecular biology?

  • Enhancing the activity of Taq polymerase
  • Inhibiting the PCR process
  • Binding to double stranded DNA and increasing fluorescence (correct)
  • Binding to single stranded RNA

    • The TaqMan probe fluoresces continuously as it hybridizes to the target gene.

    False (B)

    What does the Ct value represent in quantitative real-time PCR?

    The cycle threshold at which the amplification plot crosses a predefined threshold

    The __________ dye is used in QPCR for intercalating with double stranded DNA.

    <p>SYBR Green I</p> Signup and view all the answers

    Match the following terms with their correct descriptions:

    <p>SYBR Green I = Binds all double stranded DNA, increasing fluorescence TaqMan probe = Target specific gene with dual labeling of dye and quencher 5' nuclease activity = Cleaves the TaqMan probe during PCR Ct value = The cycle threshold at which amplification is detected</p> Signup and view all the answers

    What happens to the TaqMan probe during each PCR cycle?

    <p>It is cleaved, and the dye is released to fluoresce (A)</p> Signup and view all the answers

    The amplification plot in QPCR can provide real-time insights into the PCR process.

    <p>True (A)</p> Signup and view all the answers

    What role does the quencher play in relation to the TaqMan probe?

    <p>It prevents the dye from fluorescing while the probe is intact.</p> Signup and view all the answers

    What is the primary purpose of using a radioactive or fluorescent probe in Northern blotting?

    <p>To confirm the presence and size of specific RNA transcripts (C)</p> Signup and view all the answers

    Northern blotting provides a verifiable method for determining RNA expression patterns.

    <p>True (A)</p> Signup and view all the answers

    What technique is used to separate RNA molecules before transferring them to a membrane?

    <p>Gel electrophoresis</p> Signup and view all the answers

    In Northern blotting, the transferred RNA is immobilized on a ________ membrane.

    <p>nitrocellulose</p> Signup and view all the answers

    Match the following components with their roles in Northern blotting:

    <p>Probe = Complimentary to the gene of interest Gel electrophoresis = Separates RNA by size Nitrocellulose membrane = Holds the immobilized RNA Radiolabeling = Visualizes specific RNA transcripts</p> Signup and view all the answers

    Which of the following is a challenge associated with Northern blotting?

    <p>Time-consuming and complex (D)</p> Signup and view all the answers

    What is cDNA library primarily made from?

    <p>mRNA extracted from a cell (C)</p> Signup and view all the answers

    CDNA libraries represent all genes expressed in a sample equally.

    <p>False (B)</p> Signup and view all the answers

    What are the two main methods for measuring gene expression mentioned?

    <p>Gene expression microarrays and RNA sequencing</p> Signup and view all the answers

    CDNA is more stable than storing __________.

    <p>mRNA</p> Signup and view all the answers

    Match the method to its description:

    <p>Gene expression microarrays = Uses DNA probes on a chip to detect cDNA RNA sequencing = High throughput sequencing of cDNA libraries cDNA library = Collection of cDNAs made from mRNA Plasmid vectors = Used for bacterial cloning of cDNA</p> Signup and view all the answers

    Which process allows for the amplification of cDNA libraries?

    <p>Bacterial cloning (B)</p> Signup and view all the answers

    Gene expression microarrays can be used to compare the expression of many genes between samples.

    <p>True (A)</p> Signup and view all the answers

    What is the significance of fluorescent signals in gene expression microarrays?

    <p>They indicate hybridization of cDNA to probes on the array.</p> Signup and view all the answers

    What is the primary purpose of subtractive library screening?

    <p>To identify genes that are differently expressed between two tissues (A)</p> Signup and view all the answers

    Subtractive library screening does not involve the use of cDNAs.

    <p>False (B)</p> Signup and view all the answers

    What technique can be used to amplify unique genes after subtractive library screening?

    <p>PCR</p> Signup and view all the answers

    In subtractive library screening, cDNAs from the tissue of interest have __________ added.

    <p>adapters</p> Signup and view all the answers

    Match the terms related to subtractive library screening with their descriptions:

    <p>cDNA = Complementary DNA synthesized from mRNA Adapters = Short, known sequences added to cDNAs Subtraction = Removal of similar genes from analysis Differential gene expression = Variation in gene expression between different tissues</p> Signup and view all the answers

    What happens to similar genes during subtractive library screening?

    <p>They are subtracted (D)</p> Signup and view all the answers

    Subtractive library screening is a recent method for gene expression analysis.

    <p>False (B)</p> Signup and view all the answers

    How many different tissues were involved in the dot blot hybridization experiment mentioned?

    <p>Three</p> Signup and view all the answers

    What is the purpose of the standard curve in absolute QPCR?

    <p>To infer the absolute quantity of an unknown sample (A)</p> Signup and view all the answers

    Absolute QPCR requires co-amplification of a housekeeping gene for every sample.

    <p>False (B)</p> Signup and view all the answers

    What is calculated from each sample in the serial dilution during QPCR?

    <p>Ct value</p> Signup and view all the answers

    The process of diluting from 10^8 to __________ copies is termed serial dilution.

    <p>10^1</p> Signup and view all the answers

    Match the following terms with their descriptions:

    <p>Ct value = The threshold cycle number at which the fluorescence of a PCR product is detected Serial dilution = A method of reducing the concentration of a substance by a known factor Standard curve = A graphical representation used to estimate the quantity of an unknown sample QPCR = A quantitative PCR technique used to amplify and quantify DNA</p> Signup and view all the answers

    Which of the following statements is considered a disadvantage of absolute QPCR?

    <p>You need a previously quantified cDNA to set up a standard curve (D)</p> Signup and view all the answers

    Unknown samples and standard curve samples are typically analyzed at different times.

    <p>False (B)</p> Signup and view all the answers

    Describe one advantage of using the absolute QPCR method.

    <p>You do not need to co-amplify a housekeeping gene for every sample.</p> Signup and view all the answers

    Study Notes

    Northern Blotting

    • RNA immobilized on a membrane is incubated with a radioactive or fluorescent probe complementary to the gene of interest.
    • Visualization of the probe indicates the presence and size of RNA using a size marker or ladder.
    • A Northern blot can confirm specific gene expression patterns, as exemplified by Riordan et al.'s analysis of CFTR in 1989.
    • Gel electrophoresis of total RNA, termed 'Northern,’ transfers RNA from gel to a nitrocellulose membrane, facilitating specific transcript detection.
    • Northern blotting demonstrates relative quantities and sizes of specific RNA transcripts but is time-consuming and requires substantial RNA input.

    Fluorescent Dyes and Probes

    • SYBR Green I is a cyanine dye that intercalates with double-stranded DNA, increasing fluorescence up to 1,000-fold upon binding.
    • Taqman probes are dual-labeled with a dye and quencher, designed for specific hybridization to PCR products.
    • During PCR cycles, the Taq polymerase cleaves the probe, releasing the dye and allowing fluorescence which increases with PCR product accumulation.

    Quantitative Real-Time PCR (QPCR)

    • QPCR generates real-time amplification plots, with the point where the plot crosses a threshold known as the Ct value (cycle threshold).
    • A standard curve enables estimation of unknown sample quantities based on their Ct values.
    • Ct values from serial dilutions (e.g., 10^8 to 10^1 copies) are plotted to create a standard curve for quantification.

    Advantages and Disadvantages of QPCR

    • An advantage of absolute QPCR is the elimination of the need to co-amplify housekeeping genes for every sample.
    • A disadvantage includes requiring previously quantified cDNA for setting up standard curves.

    Subtractive Library Screening

    • This method detects differentially expressed genes between two tissues or states by mixing cDNAs from target and control tissues.
    • It involves adding adapters to cDNAs of interest and subtracting similar genes to isolate unique genes.
    • Subtractive screening serves as an early technique for analyzing differential gene expression (DGE).

    cDNA Libraries

    • cDNA libraries consist of cDNAs obtained from mRNA of a specific cell, tissue, or organism, providing a stable representation of the transcriptome during sampling.
    • These libraries can be amplified via bacterial cloning with plasmid vectors, and are utilized in differential gene expression analysis.
    • The cloned cDNA library typically reflects only the most highly expressed genes.

    Large-Scale Genomics for Gene Expression

    • Two primary technologies for measuring gene expression include gene expression microarrays and RNA sequencing.
    • Gene expression microarrays feature thousands of DNA probes corresponding to all known genes, fixed to a solid surface for hybridization with cDNA, generating fluorescent signals.
    • RNA sequencing involves isolating RNAs, preparing libraries by fragmenting and generating cDNA, and performing high-throughput sequencing for analysis.

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    Related Documents

    L10 - Methods for Measuring Gene Expression KEATS PDF

    Description

    This quiz covers the principles and techniques of Northern blotting, focusing on the visualization of RNA through probes. Learn how to interpret results, including RNA size and presence as shown through various imaging methods, and the importance of size markers. Perfect for students in genetics courses.

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