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    -DNA is very long so to get a copy of the DNA fragment which contains gene you are interested in, one of the 2 methods have to be used, reverse transcriptase and restriction endonucleases

    I understand how the reverse transcriptase method can get DNA but am unsure about endonucleases. I understand that restriction endonucleases cut up DNA but dont see how this could find the DNA fragment you are trying to isolate. Can anyone explain this please?

    After getting the fragment, its inserted into a vector using the same endonuclease enzyme used to obtain the fragment. What happens if the method used to obtain the fragments was using reverse transcriptase? does the endonuclease enzyme still have to be used

    finally, is polymerase chain reaction a way of isolating the gene, amplifying the gene or both?
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    (Original post by helpme456)
    -DNA is very long so to get a copy of the DNA fragment which contains gene you are interested in, one of the 2 methods have to be used, reverse transcriptase and restriction endonucleases

    I understand how the reverse transcriptase method can get DNA but am unsure about endonucleases. I understand that restriction endonucleases cut up DNA but dont see how this could find the DNA fragment you are trying to isolate. Can anyone explain this please?

    After getting the fragment, its inserted into a vector using the same endonuclease enzyme used to obtain the fragment. What happens if the method used to obtain the fragments was using reverse transcriptase? does the endonuclease enzyme still have to be used
    Try researching Gel electrophoresis or maybe PCR. It may be what you're looking for?

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    (Original post by helpme456)
    -DNA is very long so to get a copy of the DNA fragment which contains gene you are interested in, one of the 2 methods have to be used, reverse transcriptase and restriction endonucleases

    I understand how the reverse transcriptase method can get DNA but am unsure about endonucleases. I understand that restriction endonucleases cut up DNA but dont see how this could find the DNA fragment you are trying to isolate. Can anyone explain this please?

    After getting the fragment, its inserted into a vector using the same endonuclease enzyme used to obtain the fragment. What happens if the method used to obtain the fragments was using reverse transcriptase? does the endonuclease enzyme still have to be used

    finally, is polymerase chain reaction a way of isolating the gene, amplifying the gene or both?
    Restriction endonucleases cut the DNA at special flanking sites characterized--not everytime-- by palindromic sequences. They also cut the plasmid with corresponding sticky ends to get the gene in the vector.

    PCR is used for amplifying the gene only. The name itself proposes this.
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    (Original post by Dynamo123)
    Restriction endonucleases cut the DNA at special flanking sites characterized--not everytime-- by palindromic sequences. They also cut the plasmid with corresponding sticky ends to get the gene in the vector.

    PCR is used for amplifying the gene only. The name itself proposes this.
    what happens if you get the DNA fragments from reverse transcriptase. Is the only way of amplifying it through PCR or can you use the vector method.

    If you can use the vector method, how is the DNA joined to the plasmid.
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    DNA is joined to plasmid by another enzyme called DNA Ligase that seals the ends at sticky sites.
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    (Original post by helpme456)
    -DNA is very long so to get a copy of the DNA fragment which contains gene you are interested in, one of the 2 methods have to be used, reverse transcriptase and restriction endonucleases
    I understand how the reverse transcriptase method can get DNA but am unsure about endonucleases. I understand that restriction endonucleases cut up DNA but dont see how this could find the DNA fragment you are trying to isolate. Can anyone explain this please?

    After getting the fragment, its inserted into a vector using the same endonuclease enzyme used to obtain the fragment. What happens if the method used to obtain the fragments was using reverse transcriptase? does the endonuclease enzyme still have to be used

    finally, is polymerase chain reaction a way of isolating the gene, amplifying the gene or both?
    Where did you get this information from?

    For vector ligation, you would usually amplify the DNA that you wanted before restriction digestion. You could amplify the DNA fragment directly by PCR, or could alternatively reverse-transcribe the transcript and then amplify it (RT-PCR).

    For normal PCR, both forward and reverse primers are created such that they contain specific restriction sites on the 5' end. This means that, following amplification, you would get a DNA product with restriction sites at both ends.

    For RT-PCR, you would first generate a cDNA product with blunt ends. You would then have to ligate oligonucleotide linkers to both ends of the cDNA product using a high concentration of DNA ligase. You would design the oligonucelotides to contain restriction sites, so that you would generate restriction sites at both ends following ligation to the cDNA.

    After this, you would simply digest your DNA product and vector with the same restriction enzymes, and then ligate both together.
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    (Original post by Ashnard)
    Where did you get this information from?

    For vector ligation, you would usually amplify the DNA that you wanted before restriction digestion. You could amplify the DNA fragment directly by PCR, or could alternatively reverse-transcribe the transcript and then amplify it (RT-PCR).

    For normal PCR, both forward and reverse primers are created such that they contain specific restriction sites on the 5' end. This means that, following amplification, you would get a DNA product with restriction sites at both ends.

    For RT-PCR, you would first generate a cDNA product with blunt ends. You would then have to ligate oligonucleotide linkers to both ends of the cDNA product using a high concentration of DNA ligase. You would design the oligonucelotides to contain restriction sites, so that you would generate restriction sites at both ends following ligation to the cDNA.

    After this, you would simply digest your DNA product and vector with the same restriction enzymes, and then ligate both together.
    This has completely thrown me. Can you go a break it down a bit please

    so there are 2 steps involved in gene cloning, Isolation and amplification.

    2 ways of isolation are reverse transcriptase and restriction endonucleases

    2 ways of amplification are in vivo and in vitro:

    1.In vivo involves using vector ligation where we use a plasmid, insert into cell and use florescence to see if it has been taken in.
    2.In Vitro involves the PCR
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    (Original post by Dynamo123)
    DNA is joined to plasmid by another enzyme called DNA Ligase that seals the ends at sticky sites.
    But cDNA doesnt have sticky sites. does this mean restriction endonucleases have to be used after reverse transcriptase if you want to do vector ligation
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    (Original post by helpme456)
    This has completely thrown me. Can you go a break it down a bit please

    so there are 2 steps involved in gene cloning, Isolation and amplification.

    2 ways of isolation are reverse transcriptase and restriction endonucleases

    2 ways of amplification are in vivo and in vitro:

    1.In vivo involves using vector ligation where we use a plasmid, insert into cell and use florescence to see if it has been taken in.
    2.In Vitro involves the PCR
    Okay. I perhaps gave you more detail than you needed so you can just forget most of what I said.

    The main point (from your perspective) is how to digest the reverse transcribed DNA. Reverse transcribed DNA has blunt ends. So what you need to do first is ligate oligonucleotide linker DNA at both ends of the reverse transcribed DNA (i.e. cDNA) using a high concentration of ligase. The oligonucleotide linkers will contain restriction sites themselves, so when you ligate them to the end of the cDNA, you will then be able to restriction digest the cDNA and then insert it into the digested vector.
 
 
 
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