[Nescafe]

Hi,

I am confused with regards to a few things.

1) Are plasmids double stranded or single stranded or they can be either? I always thought they were single stranded and the reason I say this is because I always saw pictures where they would show a three codon sequence (e.g. TGA) on the plasmid that encoded amino acid XXX and then they would do a site directed mutagensis and replace it with a stop codon (TAG - UAG on the mRNA).

2) Lets say it is double stranded, how do you actually go about doing site directed mutagenesis on a three codon sequence on a double stranded DNA? Do we denature the plasmid, ligate it with a mutated sequence and do PCR? How would you even know where this sequence is, what it encodes etc. Text books make it seem like magic but I want to know the details behind how we know all this.

Thanks,

Nescafe.

[Arkcon]

Bacterial plasmids are double stranded.  One strand is coding, and the other is not.  IIRC, all genes are on the same strand, but I could be wrong, that could be for Eukaryota only.  Or you know, wrong in rare instances for all living things.  Or never right, YMMV.

For ligating in a gene, then yes, you will denature, cut with an endonuclease specific for where you want to ligate your gene.  If the plasmid doesn't end up too big, the bacterial polymerase will rebuild the complementary strand.   You will have to know something about the sequence of the plasmid, maybe by partially sequencing it, or by trying random endonucleases, and recording what works.  It does seem a little bit magic in the textbook, but a lot of this work has already been done.

[Babcock_Hall]

One way to perform PCR-based mutagenesis is to create a deliberate mismatch in one or both of the primers.  There is also Kunkel mutagenesis:  http://www.bio.davidson.edu/courses/molbio/kunkel/kunkel.html
Original citation:  http://www.pnas.org/content/82/2/488.abstract

[Yggdrasil]

Bacterial plasmids are double stranded.  One strand is coding, and the other is not.  IIRC, all genes are on the same strand, but I could be wrong, that could be for Eukaryota only.  Or you know, wrong in rare instances for all living things.  Or never right, YMMV.

Genes can be on different strands in any domain of life (bacteria, archaea or eukarya).


For PCR-based site directed mutagenesis, page 2 of the following manual has a nice diagram showing how the process works (http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf).  You denature the two strands, use PCR primers containing the mutation you want to introduce to copy the two strands, then remove the original, unmutated plasmid (no ligation required as the bacteria themselves will fix the nicks once you introduce the mutated plasmid into the bacteria).

Most of the time when performing the procedure, you would have sequenced the plasmid beforehand, so you know which codons code for the different amino acids of your protein.

[Arkcon]

Bacterial plasmids are double stranded.  One strand is coding, and the other is not.  IIRC, all genes are on the same strand, but I could be wrong, that could be for Eukaryota only.  Or you know, wrong in rare instances for all living things.  Or never right, YMMV.

Genes can be on different strands in any domain of life (bacteria, archaea or eukarya).

Gah.  I had heard that, but had forgotten. 

[Nescafe]

Bacterial plasmids are double stranded.  One strand is coding, and the other is not.  IIRC, all genes are on the same strand, but I could be wrong, that could be for Eukaryota only.  Or you know, wrong in rare instances for all living things.  Or never right, YMMV.

Genes can be on different strands in any domain of life (bacteria, archaea or eukarya).


For PCR-based site directed mutagenesis, page 2 of the following manual has a nice diagram showing how the process works (http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf).  You denature the two strands, use PCR primers containing the mutation you want to introduce to copy the two strands, then remove the original, unmutated plasmid (no ligation required as the bacteria themselves will fix the nicks once you introduce the mutated plasmid into the bacteria).

Most of the time when performing the procedure, you would have sequenced the plasmid beforehand, so you know which codons code for the different amino acids of your protein.

You sir, know, EVERYTHING.

Thanks again,

Nescafe.

[Nescafe]

Bacterial plasmids are double stranded.  One strand is coding, and the other is not.  IIRC, all genes are on the same strand, but I could be wrong, that could be for Eukaryota only.  Or you know, wrong in rare instances for all living things.  Or never right, YMMV.

Genes can be on different strands in any domain of life (bacteria, archaea or eukarya).


For PCR-based site directed mutagenesis, page 2 of the following manual has a nice diagram showing how the process works (http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf).  You denature the two strands, use PCR primers containing the mutation you want to introduce to copy the two strands, then remove the original, unmutated plasmid (no ligation required as the bacteria themselves will fix the nicks once you introduce the mutated plasmid into the bacteria).

Most of the time when performing the procedure, you would have sequenced the plasmid beforehand, so you know which codons code for the different amino acids of your protein.

I have another question, why is the parental DNA methylated?

[Yggdrasil]

Bacteria methylate their DNA in order to allow them to detect errors in DNA replication.

Imagine that you copy a strand of DNA and produce an error.  The resulting DNA might look like this:

5' - GATCATTCG - 3'
3' - CTAGTATGC - 5'

The DNA repair proteins will easily recognize the T-T mismatch, but it's unclear which T is correct.  Is the correct sequence of the top strand GATCTTCG and the mismatch due to the incorrect incorporation of T into the bottom strand or was the T incorrectly incorporated into the top strand and the top strand should read GATCTACG?

Knowing which strand was the original strand and which strand was newly synthesized would aid in figuring out which nucleotide to repair.  This is where DNA methylation comes in.  Before DNA replication, all the bacterial DNA is methylated at certain positions throughout the bacterial chromosome.  Newly synthesized DNA, however, will not contain methylation marks because the enzymes have not had a chance yet to add the methyl groups onto the new DNA.  Therefore, after DNA replication, DNA repair enzymes can recognize the original strand as the one that contains methylation while the new strand is the strand lacking methylation.

See https://en.wikipedia.org/wiki/Dam_%28methylase%29 for more information.

[Nescafe]

So interesting, how clever is that?..

Thanks so much!

Nescafe.