Molecular Biology Techniques Quiz
42 Questions
3 Views

Choose a study mode

Play Quiz
Study Flashcards
Spaced Repetition
Chat to Lesson

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the primary purpose of using probes in the identification of clones containing a gene of interest?

  • To introduce the gene of interest into a host organism
  • To precisely cut the DNA at specific locations to isolate the gene
  • To amplify the gene of interest within the clones
  • To detect specific nucleic acid sequences by hybridization (correct)

    • Which of the following is NOT a key feature of probes used in clone identification?

  • Probes can be either single-stranded or double-stranded DNA.
  • Probes allow for the visualization of hybridized nucleic acids through autoradiography.
  • Probes can only be synthesized chemically, not through biological means. (correct)
  • The size of probes can range from a few nucleotides to hundreds of kilobases.

    • How does antibody screening differ from hybridization screening for the detection of genes?

  • Antibody screening is more efficient but less specific than hybridization screening.
  • Antibody screening targets the RNA transcript of the gene, while hybridization screening targets the gene's protein product.
  • Antibody screening directly targets the protein product of the gene, while hybridization screening focuses on the gene's DNA sequence. (correct)
  • Antibody screening uses enzymes to break down the DNA, while hybridization screening relies on PCR for amplification.

    • What is the purpose of the second antibody used in antibody screening?

    <p>To bind to the first antibody and provide a detectable signal, indicating the presence of the target protein. (D)</p> Signup and view all the answers

    What is a key advantage of PCR-based screening compared to hybridization-based screening for gene identification?

    <p>PCR-based screening is more sensitive, allowing for the detection of very small amounts of target DNA. (B)</p> Signup and view all the answers

    What is the most crucial step in constructing a cDNA library?

    <p>Isolation of mRNA (A)</p> Signup and view all the answers

    Which enzyme is responsible for synthesizing the 1st strand of cDNA using mRNA as a template?

    <p>Reverse Transcriptase (C)</p> Signup and view all the answers

    Why are oligo dT primers essential in mRNA purification?

    <p>They bind to the poly A tail of mRNA. (C)</p> Signup and view all the answers

    What is the role of S1 nuclease in cDNA construction?

    <p>It digests the hairpin loop formed during cDNA synthesis. (C)</p> Signup and view all the answers

    Why does cDNA lack introns?

    <p>cDNA is synthesized from mRNA, which lacks introns. (D)</p> Signup and view all the answers

    What is the primary difference between DNA and mRNA in eukaryotic cells?

    <p>DNA contains introns, while mRNA does not. (A)</p> Signup and view all the answers

    What is the purpose of grinding a cell mass or tissue in liquid nitrogen during RNA extraction?

    <p>To prevent RNA degradation by RNases (C)</p> Signup and view all the answers

    Why is a DEPC (diethylpyrocarbonate) treatment necessary for plasticware used in RNA extraction?

    <p>To inactivate RNases that may be present on the plastic surface (C)</p> Signup and view all the answers

    Which of the following statements accurately describes the role of DNA polymerase in cDNA construction?

    <p>It synthesizes the 2nd strand of cDNA using the 1st strand as a template. (B)</p> Signup and view all the answers

    What is the primary function of oligo(dT) columns in the purification of mRNA?

    <p>To bind to the poly-A tail of mRNA (D)</p> Signup and view all the answers

    What is the significance of the 'cap' and 'poly A tail' modifications in eukaryotic mRNA?

    <p>They protect the mRNA from degradation. (C), They help in the binding of mRNA to ribosomes for translation. (D)</p> Signup and view all the answers

    Why are cDNA libraries advantageous over genomic DNA libraries for studying gene expression?

    <p>cDNA libraries represent only the expressed regions of the genome (B)</p> Signup and view all the answers

    What is the purpose of adding linkers, adapters, or homopolymers to cDNA prior to ligation into a vector?

    <p>To facilitate the cloning of cDNA into a vector (A)</p> Signup and view all the answers

    What is the purpose of plating transformed bacterial cells on selective media after cDNA ligation?

    <p>To ensure that only cells containing the desired cDNA are selected (D)</p> Signup and view all the answers

    What is a potential application of cDNA libraries in the field of reverse genetics?

    <p>To identify the function of a specific gene by studying its expression in different cell types (C)</p> Signup and view all the answers

    Which of the following is NOT a key difference between cDNA and genomic DNA libraries?

    <p>cDNA libraries are more complex to construct and maintain than genomic DNA libraries (A)</p> Signup and view all the answers

    What is the purpose of transferring induced proteins made by phage to a filter?

    <p>To identify phage clones that express the protein of interest (B)</p> Signup and view all the answers

    Which of the following methods is used to label a probe for colony hybridization?

    <p>Using a random hexamer and fill-in reaction with Klenow fragment (A)</p> Signup and view all the answers

    In the colony hybridization technique, what is the purpose of lysing the bacteria and denaturing the DNA?

    <p>To make the DNA accessible to the radiolabelled probe (A)</p> Signup and view all the answers

    In the context of screening a recombinant library, what is the purpose of using a probe that is complementary to the cloned gene?

    <p>To identify the specific clone containing the gene of interest (C)</p> Signup and view all the answers

    Why is it important for a phage clone to represent a copy of an mRNA from the cells used to make the cDNA library?

    <p>To ensure the phage clone encodes the correct protein (C)</p> Signup and view all the answers

    Which of these methods is NOT used to prepare a genomic library?

    <p>Reverse transcriptase (C)</p> Signup and view all the answers

    Which type of vector would be most suitable for cloning a genomic DNA fragment of approximately 500 kb?

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

    What is the purpose of dephosphorylation in genomic library construction?

    <p>To prevent self-ligation of the vector (B)</p> Signup and view all the answers

    Which of the following is NOT a method for introducing recombinant DNA into host cells for genomic library construction?

    <p>Microinjection (D)</p> Signup and view all the answers

    Which of these techniques is commonly used for screening a genomic library?

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

    What is the primary advantage of using a genomic library over a cDNA library?

    <p>Genomic libraries represent the entire genome, including non-coding regions. (A)</p> Signup and view all the answers

    What is the purpose of using selective agar plates when plating transformed cells in genomic library construction?

    <p>To inhibit the growth of untransformed cells (C)</p> Signup and view all the answers

    Which of the following is NOT a potential application of a genomic library?

    <p>Studying protein-protein interactions (D)</p> Signup and view all the answers

    What is the primary challenge in isolating a specific gene from a cell's DNA directly?

    <p>The gene is present in very small amounts compared to other DNA sequences. (A)</p> Signup and view all the answers

    What is the main reason for using partial digestion with restriction enzymes when constructing a genomic library?

    <p>To increase the chances of finding intact genes within the fragmented DNA. (B)</p> Signup and view all the answers

    Which of the following vectors offers the capacity to incorporate the largest DNA segments?

    <p>Yeast Artificial Chromosomes (YACs) (C)</p> Signup and view all the answers

    The term 'shotgun cloning' is used to describe the process of genomic library construction because:

    <p>The process involves randomly inserting DNA fragments into vectors. (A)</p> Signup and view all the answers

    What is the primary difference between a genomic library and a cDNA library?

    <p>A genomic library represents the entire genome, while a cDNA library only represents the genes expressed in a specific cell type. (D)</p> Signup and view all the answers

    In the construction of a genomic library, why is it important to select DNA fragments between 10-20 kb in size?

    <p>This size range ensures that the fragments are large enough to contain complete genes. (C)</p> Signup and view all the answers

    What role does a gene library play in identifying a specific gene of interest?

    <p>It serves as a source of DNA fragments, allowing researchers to screen for the desired gene. (A)</p> Signup and view all the answers

    Which of the following is NOT a step involved in constructing a genomic library?

    <p>Sequencing of the entire genome of the target organism. (B)</p> Signup and view all the answers

    Flashcards

    DNA Library

    A collection of DNA clones representing an organism's genome or expressed genes.

    Gene Library

    Cloned DNA fragments that represent the genes of a specific organism.

    Shotgun Cloning

    A method of cloning all the genes of an organism without targeting specific ones.

    Genomic Library Construction

    The process of creating a genomic library by isolating and cloning DNA fragments.

    Signup and view all the flashcards

    Restriction Enzymes

    Proteins that cut DNA at specific sequences, used in genomic library construction.

    Signup and view all the flashcards

    cDNA Library

    A library composed of complementary DNA, representing genes expressed in a cell type.

    Signup and view all the flashcards

    Gene Bank

    Another term for a gene library, where cloned DNA fragments are stored.

    Signup and view all the flashcards

    Vectors in Cloning

    Molecules like plasmids or viruses that carry DNA fragments into host cells for cloning.

    Signup and view all the flashcards

    Isolate Genomic DNA

    Extract genomic DNA from the organism and ensure its quality through methods like phenol-chloroform extraction and ethanol precipitation.

    Signup and view all the flashcards

    Fragmentation

    Use restriction enzymes or mechanical shearing to cut genomic DNA into smaller, manageable fragments.

    Signup and view all the flashcards

    Prepare the Vector

    Select a suitable vector based on fragment size and digest it with restriction enzymes for ligation.

    Signup and view all the flashcards

    Ligate DNA Fragments

    Join genomic DNA fragments to the vector using DNA ligase and ATP to form recombinant DNA.

    Signup and view all the flashcards

    Transform Host Cells

    Introduce recombinant DNA into a host organism such as E. coli using methods like heat shock or electroporation.

    Signup and view all the flashcards

    Plate the Transformants

    Spread transformed cells on selective agar plates to culture and select successful transformants.

    Signup and view all the flashcards

    Screen the Library

    Use methods like colony hybridization and PCR-based screening to identify specific DNA sequences in the library.

    Signup and view all the flashcards

    Store and Maintain the Library

    Preserve the genomic library as glycerol stocks at low temperatures for long-term use.

    Signup and view all the flashcards

    cDNA

    Complementary DNA synthesized from mRNA; represents expressed genes.

    Signup and view all the flashcards

    Intron

    Non-coding sequences in DNA that are removed from mRNA during splicing.

    Signup and view all the flashcards

    Exon

    Coding sequences in DNA that remain in mRNA after splicing.

    Signup and view all the flashcards

    Splicing

    Process of removing introns and joining exons in mRNA.

    Signup and view all the flashcards

    Reverse Transcriptase

    Enzyme that synthesizes the first strand of cDNA using mRNA as a template.

    Signup and view all the flashcards

    mRNA Isolation

    Process of extracting mRNA to ensure quality for cDNA synthesis.

    Signup and view all the flashcards

    Oligo dT Primers

    Short DNA sequences that bind to the poly A tail of mRNA, aiding in purification.

    Signup and view all the flashcards

    DNA Polymerase

    Enzyme that synthesizes the second strand of cDNA using the first strand as a template.

    Signup and view all the flashcards

    Gene Clone Identification

    The process of finding specific clones with a desired gene from a large pool of clones.

    Signup and view all the flashcards

    DNA Probe

    A short piece of DNA or RNA used to detect specific nucleic acid sequences through hybridization.

    Signup and view all the flashcards

    Hybridization

    The binding of two nucleic acid chains by base pairing during detection processes.

    Signup and view all the flashcards

    Antibody Screening

    A method of identifying clones by using antibodies that recognize and bind to specific proteins.

    Signup and view all the flashcards

    Secondary Antibody

    An antibody used to detect the primary antibody, often labeled for identification purposes.

    Signup and view all the flashcards

    λ Library Construction

    A method to express proteins encoded by cDNA in phage.

    Signup and view all the flashcards

    Colony Hybridization

    A technique to screen plasmid libraries using specific probes.

    Signup and view all the flashcards

    Probe Labeling Method I

    Uses random hexamer and Klenow fragment for probe labeling.

    Signup and view all the flashcards

    Probe Labeling Method II

    Uses T7 polynucleotide kinase for end labeling of probes.

    Signup and view all the flashcards

    Filtering Protein Antibodies

    Screening plaques with antibodies to identify specific proteins.

    Signup and view all the flashcards

    DEPC Treatment

    All plasticware must be treated with diethylpyrocarbonate to eliminate RNAses.

    Signup and view all the flashcards

    RNA Extraction

    Process of isolating RNA from cells or tissue, requiring precautions to prevent RNAse contamination.

    Signup and view all the flashcards

    mRNA Purification

    Isolate mRNA by removing rRNA and tRNA using Oligo(dT) columns.

    Signup and view all the flashcards

    Vector Ligation

    Adding linkers or adapters to cDNA before introducing it into a bacterial host.

    Signup and view all the flashcards

    cDNA vs. Genomic Library

    cDNA libraries lack non-coding regions, while genomic libraries provide both coding and non-coding sequences.

    Signup and view all the flashcards

    Selection Media

    Nutrient environment used to grow transformed bacteria, selecting for successful transformation.

    Signup and view all the flashcards

    Screening Gene Library

    Identifying specific bacterial or phage clones that contain desired genes in a genomic library.

    Signup and view all the flashcards

    Study Notes

    DNA Libraries and Screening for Recombinants

    • A DNA library is a collection of DNA clones representing either the entire genome of an organism or a set of genes expressed in a specific cell type.
    • Gene libraries are sets of cloned DNA fragments representing the genes of a specific organism.
    • Isolating specific genes directly from a genome is impractical due to the large quantity of extraneous DNA.
    • Genomic libraries are made by isolating total DNA, digesting it into suitable fragments, and cloning those fragments into appropriate vectors.
    • Shotgun cloning is a strategy to clone all genes in an organism without targeting specific genes.
    • Recombinant clones are screened to find those containing the desired gene.
    • DNA is frequently partially digested to ensure that not every restriction site is cleaved in every DNA molecule.
    • Overlapping fragments allow the reconstruction of the original DNA sequence.

    Construction of Genomic Libraries

    • Isolate Genomic DNA: Extract DNA from the organism of interest, ensuring high quality through methods like phenol-chloroform extraction and ethanol precipitation. Quantify and check purity using spectrophotometry or gel electrophoresis.
    • Fragmentation: Digest DNA into manageable fragments using restriction enzymes or mechanical shearing (like sonication).
    • Size Selection: Use agarose gel electrophoresis to select the DNA fragments of the desired size range, then extract using gel extraction methods.

    Prepare the Vector

    • Choose vectors appropriate for fragment sizes (plasmids, lambda phage vectors, BACs or YACs).
    • Digest the vector using compatible restriction enzymes, opening it at the appropriate site.
    • Dephosphorylate the vector to prevent self-ligation.

    Ligate DNA Fragments into the Vector

    • Mix the fragmented genomic DNA with the prepared vector in the presence of DNA ligase and ATP.
    • DNA ligase joins the DNA fragments to the vector, creating recombinant DNA.

    Transform Host Cells

    • Introduce the recombinant DNA into a host organism (e.g., E. coli) using methods like heat shock, electroporation or in vitro packaging.

    Plate the Transformants

    • Spread the transformed cells on selective agar plates containing antibiotics or other markers to select for successful transformants.

    Screen the Library

    • Colony Hybridization: Use labeled probes to detect colonies containing specific DNA sequences.
    • PCR-Based Screening: Amplify target sequences to identify desired clones.
    • Store the genomic library as glycerol stocks at -80°C or as DNA libraries in appropriate buffers for long-term use.

    Applications of Genomic Libraries

    • Identifying genes and regulatory elements.
    • Studying genetic disorders and mutations.
    • Sequencing entire genomes.
    • Producing recombinant protein or transgenic organisms

    cDNA Cloning

    • A cDNA library is a collection of DNA clones representing expressed genes in a specific cell or organism.
    • cDNA is made from completely transcribed mRNA.
    • cDNA libraries contain only expressed genes, lacking non-coding regions.
    • They're useful for studying expressed genes and generating protein products.

    Construction of cDNA Libraries

    • Isolating mRNA: Isolate mRNA (most crucial step). All components should be super clean and free of chromosomal DNA, RNases and phenolics.
    • Synthesis of 1st strand: Synthesis of cDNA using reverse transcriptase based on an mRNA template.
    • Synthesis of 2nd strand: Synthesis of second cDNA strand using DNA polymerase.

    Key Molecules in cDNA Construction

    • Reverse transcriptase (RTase): Synthesizes the first cDNA strand using mRNA as a template.
    • Oligo dT primers: Essential for mRNA purification.
    • DNA Polymerases: Synthesize the second cDNA strand using the first strand as a template.
    • S1 nuclease: Digests hairpin loops.

    Total RNA and mRNA Isolation

    • mRNA purity is crucial for cDNA synthesis success.
    • Use DEPC-treated materials and bake glassware to prevent RNAse contamination.
    • Grinding tissue in liquid nitrogen helps inhibit RNAse activity.

    Purify the mRNA

    • Use matrix columns containing oligo (dT) to isolate pure mRNA from total RNA.

    cDNA Library Screening Methods

    • Methods: Hybridization-based screening, PCR-based screening, and immunological screening.
    • Probes: Pieces of DNA or RNA used to detect specific nucleic acid sequences via hybridization (binding of complementary nucleic acids).

    Antibody Screening

    • Identify translation products indirectly by raising antibodies specific to purified products.
    • Screening procedure is similar to hybridization-based screening.

    Screening of Libraries

    • Probes can be labelled radioactively to detect hybridized nucleic acid via autoradiography.

    • Probes vary in size from a few nucleotides to hundreds of kilobases.

    • Initially, a probe may be double-stranded which must be single-stranded for screening purposes.

    Studying That Suits You

    Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

    Quiz Team

    Related Documents

    DNA Libraries and Screening for Recombinants PDF

    Description

    Test your knowledge on crucial molecular biology techniques like gene identification, cDNA library construction, and the use of probes and antibodies. This quiz covers key concepts including PCR screening, RNA extraction, and the differences between DNA and mRNA. Enhance your understanding of these essential methods in modern genetics.

    More Like This

    Use Quizgecko on...
    Browser
    Open
    Browser
    Browser