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    hi

    1) if two restriction enzymes produce the same sticky ends can these anneal together? (technically they should, but everywhere is says only 'same' restriction enzymes' sticky ends may anneal)


    2) if a restriction enzyme produces sticky ends already do you need to 'add lengths of single-stranded DNA using enzymes' (that's what my bio textbook says but it doesn't make sense to me!

    thanks a lot

    alaska
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    (Original post by alaskadish)

    1) if two restriction enzymes produce the same sticky ends can these anneal together? (technically they should, but everywhere is says only 'same' restriction enzymes' sticky ends may anneal)
    Yes you're right. They could anneal together.

    (Original post by alaskadish)
    2) if a restriction enzyme produces sticky ends already do you need to 'add lengths of single-stranded DNA using enzymes' (that's what my bio textbook says but it doesn't make sense to me!
    Enzymes ligate the fragments together. Even if a single stranded piece of DNA is complementary to the sticky end it won't join without joining enzymes (e.g. ligase). I think that's what your book means.

    Hope that helps :-)
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    (Original post by grovichik)
    Yes you're right. They could anneal together.



    Enzymes ligate the fragments together. Even if a single stranded piece of DNA is complementary to the sticky end it won't join without joining enzymes (e.g. ligase). I think that's what your book means.

    Hope that helps :-)
    1) thanks

    2) Yeah, that's DNA ligase for the sugar-phosphate backbone thing...what i meant was that to PRODUCE sticky ends do you need to add them or restriction enzymes already produce them or both? i suppose when restriction enzymes produce blunt ends, you would need to add sticky ends but my bio textbook for the same example first says 'the insulin genes were given sticky ends. this was done by adding lengths of single-stranded DNA made of guanine nucleotides to each end, using enzymes' and then says in the acocmpanying picture 'mRNA is taken from beta cells aand used to make DNA with the help of reverse transcriptase. It is then cut to leave sticky ends' and i was confused...do they do both at the same time? why?

    thanks very much

    alaska
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    (Original post by alaskadish)

    my bio textbook for the same example first says 'the insulin genes were given sticky ends. this was done by adding lengths of single-stranded DNA made of guanine nucleotides to each end, using enzymes'
    This is probably to insert an insulin gene into a plasmid for it to be replicated? Sticky ends need to be added so it can attach to the sticky ends on the plasmid (produced by the restriction enzymes).

    (Original post by alaskadish)
    and then says in the acocmpanying picture 'mRNA is taken from beta cells aand used to make DNA with the help of reverse transcriptase. It is then cut to leave sticky ends' and i was confused...do they do both at the same time? why?
    I'm not sure what this bit is about - what are the 'beta cells'? Is this the step before. I.e. they take the mRNA and turn it into a DNA strand which the insulin gene is then inserted into?
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    (Original post by grovichik)
    This is probably to insert an insulin gene into a plasmid for it to be replicated? Sticky ends need to be added so it can attach to the sticky ends on the plasmid (produced by the restriction enzymes).
    yes, the sticky ends are for that...but why would you need to add sticky ends if the restriction enzyme already produces them (as it says in the accompanying picture)?

    (Original post by grovichik)
    I'm not sure what this bit is about - what are the 'beta cells'? Is this the step before. I.e. they take the mRNA and turn it into a DNA strand which the insulin gene is then inserted into?
    yeah, that part is the step before...just like I said above is it cut to leave sticky ends or do you add sticky ends? you could do either I suppose but why would you do both at the same time (like my book implies)?

    thanks a lot

    PS. I have more questions so should I make another thread or can I just keep adding them on here? I'm new :o:
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    (Original post by alaskadish)
    yes, the sticky ends are for that...but why would you need to add sticky ends if the restriction enzyme already produces them (as it says in the accompanying picture)?


    yeah, that part is the step before...just like I said above is it cut to leave sticky ends or do you add sticky ends? you could do either I suppose but why would you do both at the same time (like my book implies)?

    thanks a lot

    PS. I have more questions so should I make another thread or can I just keep adding them on here? I'm new :o:
    Yep you're thinking along the right lines. It doesn't make much sense to do both. Unless the sticky ends produced by cutting out the insulin gene are not complementary to the sticky ends of the plasmid that you want to put the insulin gene into, in which case you would add different sticky ends on. That's the only explanation I can think of...

    If it's about the same topic then ask them here but if not make a new thread.

    :-)
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    (Original post by grovichik)
    Yep you're thinking along the right lines. It doesn't make much sense to do both. Unless the sticky ends produced by cutting out the insulin gene are not complementary to the sticky ends of the plasmid that you want to put the insulin gene into, in which case you would add different sticky ends on. That's the only explanation I can think of...

    If it's about the same topic then ask them here but if not make a new thread.

    :-)
    that sounds plausible...thanks!

    thank you
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    okay...one more question on the same topic

    DNA sequencing
    1) you add primers to the DNA which have complementary codes to the start of the gene....doesn't that mean you would have to know some of the gene sequence???? so what's the sense of DNA sequencing?
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    (Original post by alaskadish)
    okay...one more question on the same topic

    DNA sequencing
    1) you add primers to the DNA which have complementary codes to the start of the gene....doesn't that mean you would have to know some of the gene sequence???? so what's the sense of DNA sequencing?
    So you would have a double strand of DNA that you split into two single strands for DNA sequencing. Primers must be added because the enzymes that are involved in DNA sequencing (adding on bases) can only add to an existing sequence i.e. they can't start from nothing.

    I havn't done any sequencing myself but I suspect you just chuck random primers in with the mixture and see if any anneal (it's probably a bit more scientific than that and similar genes might be expected to have similar starting sequences and hence similar primers could be used).

    Once you've added the primers, yes you would know a small length of the sequence but you would be no where near knowing the full sequence and bits of DNA can be massive - this is why you need to DNA sequence it!

    :-)
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    (Original post by grovichik)
    So you would have a double strand of DNA that you split into two single strands for DNA sequencing. Primers must be added because the enzymes that are involved in DNA sequencing (adding on bases) can only add to an existing sequence i.e. they can't start from nothing.

    I havn't done any sequencing myself but I suspect you just chuck random primers in with the mixture and see if any anneal (it's probably a bit more scientific than that and similar genes might be expected to have similar starting sequences and hence similar primers could be used).

    Once you've added the primers, yes you would know a small length of the sequence but you would be no where near knowing the full sequence and bits of DNA can be massive - this is why you need to DNA sequence it!

    :-)
    thanks so much
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    okay, I'm really sorry but I have one more question....

    what are background genes? (if you type it in google, nothing decent comes up)
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    (Original post by alaskadish)
    okay, I'm really sorry but I have one more question....

    what are background genes? (if you type it in google, nothing decent comes up)
    Hmm..i've never heard that term before. sorry.
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    (Original post by grovichik)
    I havn't done any sequencing myself but I suspect you just chuck random primers in with the mixture and see if any anneal (it's probably a bit more scientific than that and similar genes might be expected to have similar starting sequences and hence similar primers could be used).
    This is pretty much it. As you astutely noticed, in order to sequence a gene you surely have to know something about its sequence - this is one of the hurdles that need to be overcome in DNA sequencing. You use a variety of techniques usually, random overlapping primers can be added in the hope they will cover the area of your gene of interest. You can also compare it to known similar genes and look for particular sequences which are common but unique to that type of gene.

    As for your sticky ends question, you would select the appropriate restriction enzyme to give you the right sticky ends. If you had blunt ends then you may want to 'create' sticky ends - but it's unusual to alter sticky ends, you would normally just find a different restriction enzyme.
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    @grovichik -
    thanks anyway! you've been a lot of help.

    (Original post by atheistwithfaith)
    This is pretty much it. As you astutely noticed, in order to sequence a gene you surely have to know something about its sequence - this is one of the hurdles that need to be overcome in DNA sequencing. You use a variety of techniques usually, random overlapping primers can be added in the hope they will cover the area of your gene of interest. You can also compare it to known similar genes and look for particular sequences which are common but unique to that type of gene.

    As for your sticky ends question, you would select the appropriate restriction enzyme to give you the right sticky ends. If you had blunt ends then you may want to 'create' sticky ends - but it's unusual to alter sticky ends, you would normally just find a different restriction enzyme.
    thank you!
 
 
 
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