Molecular Cloning explained for Beginners

00:06:09
https://www.youtube.com/watch?v=NoyeCMmP5tw

Summary

TLDRVideoen gir en grundig gjennomgang av molekylær kloning, en essensiell teknikk i genetisk forskning. Den beskriver prosessen fra identifisering av DNA-fragmenter til generering av vektorer, bruk av restriksjonsenzymer for å klippe DNA, og hvordan man lager rekombinant DNA. Det forklares også hvordan man isolerer og verifiserer klonede fragmenter ved hjelp av gel-purifisering og DNA-sekvensering. Denne teknikken er avgjørende for å forstå genetikk og utvikle nye medisinske behandlinger.

Takeaways

  • 🧬 Molekylær kloning er en grunnleggende teknikk i genetisk forskning.
  • 🔍 Før kloning må forskere identifisere spesifikke DNA-fragmenter.
  • 🔗 Vektorer, som plasmider, brukes til å sette inn DNA-sekvenser.
  • ✂️ Restriksjonsenzymer fungerer som molekylære saks for å kutte DNA.
  • ⚗️ Gel-purifisering separerer DNA-fragmenter basert på størrelse.
  • ✅ Verifisering av klonede fragmenter er viktig for nøyaktighet.
  • 🦠 Bakterier brukes som vertsceller for å replikere rekombinant DNA.
  • 📊 PCR og DNA-sekvensering bekrefter suksess i kloning.
  • 💡 Molekylær kloning bidrar til fremskritt innen medisin og bioteknologi.

Timeline

  • 00:00:00 - 00:06:09

    Molekylkloning er en grunnleggende teknikk innen genetisk forskning, brukt til å lage rekombinante DNA-molekyler og forsterke dem i vertsceller. Før kloning må forskere identifisere spesifikke DNA-fragmenter av interesse, som gener eller regulatoriske elementer. Den første fasen involverer generering av en vektor, ofte plasmider, som inneholder viktige elementer som antibiotikaresistensgener og opprinnelse for replikasjon. Restriksjonsenzymer brukes til å klippe DNA på bestemte steder, noe som skaper 'sticky ends' for å sammenføye DNA-fragmenter. Neste steg er å generere og forberede DNA-settet av interesse, som kan oppnås gjennom PCR eller syntetisk DNA. Etter at både vektoren og settet er kuttet, må de renses ved gel-purifikasjon for å isolere de ønskede fragmentene. Til slutt settes settet og vektoren sammen ved hjelp av ligase, og det resulterende rekombinante DNA-molekylet kan identifiseres og verifiseres ved hjelp av seleksjonsmarkører og teknikker som PCR og DNA-sekvensering.

Mind Map

Video Q&A

  • Hva er molekylær kloning?

    Molekylær kloning er en teknikk for å lage rekombinante DNA-molekyler og forsterke dem ved replikasjon i vertsceller.

  • Hva er en vektor i molekylær kloning?

    En vektor, eller backbone, er DNA-segmentet der DNA-sekvensen av interesse settes inn.

  • Hvordan brukes restriksjonsenzymer i kloning?

    Restriksjonsenzymer fungerer som molekylære saks som kutter DNA på spesifikke steder, noe som skaper overheng som kan brukes til å lime sammen DNA-fragmenter.

  • Hva er gel-purifisering?

    Gel-purifisering er en metode for å separere DNA-fragmenter basert på størrelse ved å bruke en gelmatrise.

  • Hvordan verifiseres klonede DNA-fragmenter?

    Klonede DNA-fragmenter verifiseres ved hjelp av teknikker som PCR og DNA-sekvensering.

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  • 00:00:00
    in the world of genetic research
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    molecular cloning stands as a
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    Cornerstone technique molecular cloning
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    is used to assemble recombinant DNA
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    molecules and amplify them by
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    replication within host cells in this
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    video we explore how scientists
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    manipulate DNA in the laboratory before
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    embarking on the cloning Journey
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    scientists must first identify the
  • 00:00:25
    specific DNA fragment they wish to clone
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    this fragment could represent Gene of
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    Interest a regulatory element such as a
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    promote region or any other sequence
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    that holds importance in their research
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    the first step is about generating the
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    vector also called Backbone in other
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    words the piece of DNA in which the
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    sequence of Interest should be inserted
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    in a later step a commonly used type of
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    vector are small circular pieces of DNA
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    naturally found in bacteria known as
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    plasmids common elements of the the
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    plasmid are an antibiotic resistance
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    Gene An Origin of replication which
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    enables replication of the plasmid
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    inside the bacterial cell and a promote
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    region that can drive gene expression
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    for the gene that will be pasted
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    Downstream of it the backbone also
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    contains different restriction sides
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    specific DNA sides where the DNA can be
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    cleaved later most restriction sides lie
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    Downstream of the promoter region to
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    manipulate or open DNA scientists employ
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    the use of restriction enzymes often
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    referred to as molecular scissors these
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    enzymes possess the remarkable ability
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    to recognize a defined short DNA
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    sequence known as the restriction site
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    and cleave DNA at those precise
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    locations this enzymatic action
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    generates fragments with sticky ANS
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    these sticky ANS serve as the glue that
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    brings together different DNA fragments
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    later once the backbone is generated
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    ated the second step is the insert
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    generation there are different ways to
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    generate the DNA sequence of interest
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    for example PCR amplification of a gene
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    from a genomic DNA sample or purchasing
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    a synthetic DNA fragment from a company
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    once the insert is generated it needs to
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    be prepared before being pasted into the
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    backbone a classical way of doing so is
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    with restriction digestion again
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    restriction enzymes can be used to gener
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    at the same overhangs as of the
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    backbone now that insert and Vector are
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    cut unfortunately one can't just throw
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    the digestion mixtures together insert
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    and backbone need to be isolated on the
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    other hand the enzymes used to digest
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    them as well as any pieces cut out of
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    them need to be discarded this can be
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    done with gel purification in gel
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    purification a voltage difference across
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    the gel metrix usually agross is used to
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    pull negatively charged DNA through the
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    gel the digested DNA and undigested
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    controls are loaded on the top of the
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    gel in Wells positioned towards the
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    cathode when the voltage is applied
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    across the gel the DNA migrates toward
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    the anode larger fragments of linearized
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    DNA migrate slower than smaller
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    linearized fragments the backbone can be
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    separated away from any inserts cut out
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    of it and the insert can be isolated and
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    separated from any overhangs cut off of
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    it via their different migration speeds
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    after running the gel for some duration
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    of time these differently sized pieces
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    will be in different locations and can
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    be cut out of the gel individually the
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    DNA can be extracted from the piece of
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    gel the last step is to assemble insert
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    and backbone here both digested DNA
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    fragments are incubated together the
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    overhangs which were generated earlier
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    using using restriction enzymes enables
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    scientists to join the DNA of Interest
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    with the vector ensuring compatibility
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    between the sticky ANS of the fragment
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    and the vector liase is added to link
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    the two fragments together this molecule
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    is now known as recombinant DNA since
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    the reaction will also contain
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    byproducts such as incorrectly assembled
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    or undigested plasmid molecules the next
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    step is to isolate a single correctly
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    assembled recombinant molecule from the
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    assembly reaction to this end bacteria
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    for example laboratory strains of eoli
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    are commonly employed as host cells due
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    to their ability to take up and
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    replicate individual recombinant DNA
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    molecules and produce substantial
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    quantities of the cloned DNA fragment to
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    identify the cells that have
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    successfully taken up the recumbant DNA
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    scientists employ selection markers such
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    as antibiotic resistance genes cells
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    that have taken up a recombinant DNA
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    harboring the resistance Gene Will
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    Survive and grow in the presence of a
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    specific antibiotic whereas
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    non-transformed cells will perish
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    because presence of the antibiotic
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    resistance Gene alone does not guarantee
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    that the recombinant DNA molecule has
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    been assembled as desired scientists
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    proceed to verify the presence and
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    integrity of the Clone DNA fragment
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    techniques such as polymerous chain
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    reaction and DNA sequencing Aid in
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    ensuring accuracy by comparing the
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    sequence of the Clone fragment with
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    known sequences scientists can confirm
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    the success of the cloning process
  • 00:05:44
    molecular cloning stands as a powerful
  • 00:05:46
    tool in the field of genetic research
  • 00:05:48
    allowing scientists to explore the
  • 00:05:50
    complexities of genetics or unveil the
  • 00:05:53
    mechanisms of diseases for
  • 00:05:54
    groundbreaking progress in medicine and
  • 00:05:57
    biotechnology check out this video here
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    might be interesting for you please
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    subscribe to the channel and leave a
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    like if the video was helpful to you
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    thanks a
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    lot
Tags
  • molekylær kloning
  • genetisk forskning
  • rekombinant DNA
  • restriksjonsenzymer
  • vektorer
  • PCR
  • gel-purifisering
  • DNA-sekvensering
  • antibiotikaresistens
  • genetisk manipulasjon