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454 sequencing


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#1 lyok

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Posted 01 April 2012 - 07:58 AM

Hallo all,

I have the following problem with 454: when the samples are prepared (adaptors put on the samples, made the DNA single stranded), the samples are then transferred to the beads.
Now my question is: if 1 DNA strand attaches to the bead, then what happens?

==> this DNA strand is bound to the bead and amplified, now after this step, what happens? The strand that you amplified breaks lose and gets bound at anohther place on the bead? Or is it the original strand (the one from your sample) that breaks lose and attaches at another place?
And how do you make sure the right strand breaks lose? Because you want all identical strans on your bead in the end.. you need to make sure that its the correct strand that breaks lose everytime, how do you do this?

#2 pDNA

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Posted 02 April 2012 - 12:17 PM

i'm not a specialist on the sequencing chemistry and library preperation for the 454 technology ...as far as i remember you have two adapters and adapter B contains a 5' biotin tag for the immobilization of the DNA library (dsDNA library) on an streptavidin-coated bead. In the next step the non-biotinylated single strand is removed by striping it of using 0.125 M NaOH. These ssDNA molecules have an A adaptor on their 5′ ends and B adaptor sequences on their 3′ ends (“A-B strands”), whereas the complementary strands, which carry B adaptors on their 5′ ends and A adaptors on their 3′ ends (“B-A strands”) will remain on the beads and be discarded.

Hope this answers your question?!

Best regards,
p

#3 lyok

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Posted 02 April 2012 - 01:15 PM

i'm not a specialist on the sequencing chemistry and library preperation for the 454 technology ...as far as i remember you have two adapters and adapter B contains a 5' biotin tag for the immobilization of the DNA library (dsDNA library) on an streptavidin-coated bead. In the next step the non-biotinylated single strand is removed by striping it of using 0.125 M NaOH. These ssDNA molecules have an A adaptor on their 5′ ends and B adaptor sequences on their 3′ ends (“A-B strands”), whereas the complementary strands, which carry B adaptors on their 5′ ends and A adaptors on their 3′ ends (“B-A strands”) will remain on the beads and be discarded.

Hope this answers your question?!

Best regards,
p


Ok, thanks a lot.

One more question: when you use the ssDNA molecules in the emPCR, then the beads have a short oligonucleotide sequence that helps the binding of this ssDNA to the bead and it also works as a primer.
But the second primer they use in that reaction, is this primer allready biotinylated?

I have been reading the manual, but its confusing:
(this is what the manual says about the amplification, it allready speaks of a biotinylated primer)

Amplifi cation:


The emulsifi ed beads are subjected to PCR to clonally amplify each
template DNA molecule. The DNA templates are hybridized to bead-bound oligonucleotide
primers, the capture primers (see step 2, above). These capture primers double as
PCR primers, anchoring the newly synthesized, complementary strands to the beads.
As the PCR reaction progresses, these bead-bound, complementary strands direct
the synthesis of more fi rst-strand moieties, which hybridize to an excess of bead-bound
capture primers. Also, the second, soluble amplifi cation primer is biotinylated; this
allows for enrichment of the beads carrying amplifi ed DNA later in the procedure
(see step 6, below). After amplifi cation, typical immobilized template copy number
ranges from 10 to 50 × 10


==> its says that the second primer is biotinylated, but in the rest of the manual they say that you need to add the enrichment primer (which is biotinylated) later:
(this doesnt make sense to me to be honest)

DNA Library Bead Enrichment:


The procedure above generates a certain proportion
of beads that carry no amplifi ed DNA (null beads), or little amplifi ed DNA, either because
they did not capture a molecule of template in the beginning or because the DNA
template did not amplify properly. To reduce the percentage of beads without or with
too little template, the sixth step of the procedure enriches the total bead population
for amplifi ed DNA-carrying beads. This enrichment step involves the hybridization of
the biotinylated Enrichment Primer (from the Kit) to the Adaptor “A” of each amplifi ed
template (to which it is complementary), and its binding to streptavidin-coated magnetic
beads. The bound beads (carrying amplifi ed DNA) can then be separated from the null
and poorly amplifi ed beads with a magnetic particle collector. The DNA library beads
are then separated from the magnetic beads by melting the amplifi cation products
away from the Enrichment Primer, leaving a population of bead-bound single-stranded
template DNA fragments: the immobilized and amplifi ed DNA library




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