A graphical overview of the procedure will pop-up in a new window.
 

Briefly, the process of gathering additional information about DNA inserted into the pUC19 plasmid in order to make a decision about what DNA is worth sending out to have sequenced involves 3 steps:

• Select bacterial colonies that look promising from the hybridized membranes (those that have bound a lot of the "probes" containing the sequences of interest)
• Amplify the inserted/cloned DNA using PCR primers (2 reactions, one with and one without the "probes" being used as primers) and run the products on a polyacrylamide gel
• Interpret the band patterns obtained for each pair of PCR products

Three patterns are generally visible in the side-by-side PCR product profiles where “screening oligos” were used as primers and compared to the PCR products that represent the entire length of the inserted DNA:

1. Only a shorter length fragment is produced in comparison to the entire inserted DNA fragment length.
2. Both a shorter fragment and as well as a fragment the same length as the original inserted DNA are visible.
3. Only a fragment the same length as the original total DNA insert is produced and no shorter fragment is visible.

In the first case (1) it appears that the “screening oligos” (which are simply synthesized sequences of repeated DNA) compete strongly with the sequencing primers so that the primary product is a shorter fragment that indicates the distance that the “microsatellite repeat unit” that matches one of the “screening oligos” is away from one edge of the inserted DNA sequence.

Competition for primer binding appears less pronounced when both original length and shorter fragments are produced (2).  However, in the last example (3) the “screening oligos” do not appear to bind at all, so no shorter fragment is produced.

The following diagram represents how the various primers (that initiate the DNA replication reaction) might bind to the original template DNA and produce the products that are visible as stained bands on the gels:

Locations of primer binding sites are indicated by numbers and the length of the fragment produced is represented by the bar beneath.  The lighter area beneath the “2” represents a region of DNA containing microsatellite repeat units.

“Sequencing primers” bind at points 1 and 3 produce a fragment that represents the entire length of the DNA that was previously inserted into the circular plasmid and transferred into E. coli for plasmid copy multiplication.  However, if another primer binds at a point between 1 and 3 there is potential for production of a shorter fragment as well.  These possible alternate (or additional) shorter products are represented by the lower fragments that have the microsatellite repeat as an end point.

In order for the PCR amplification reaction to occur one primer must bind to a single strand of DNA and begin replication in one direction as another primer binds to the complementary (matching) single strand of DNA and initiates replication in the opposite direction.  The products produced by the single primers moving along the complementary strands of DNA in opposite directions must overlap in order for high-copy replication to occur (see PCR page).

If one of the “screening oligos” is complementary to a sequence (2) in the larger fragment a shorter fragment may be produced, or possibly be produced as the primary product.  Below are figures representing possible simplified combinations of products produced.  Usually only one of the shorter fragments would be produced because the “screening oligos” that are added are complementary to only one strand of the DNA.  But inversions of repeat sequences or mixes of different types of repeat units can lead to the possibility of multiple short sequences.

The arrangement of the fragments visible above also represents the order they would appear in on a gel (longer fragments at the top and shorter at the bottom).  The photograph provides examples of the previous descriptions.  It is provided as a “real-world” example of how products look on gels (as opposed to a stylized diagram with perfect products for unquestionable identification).

The numbers above the paired lanes in the gel match the types of products listed at the beginning of this discussion.  “L” indicates the molecular weight ladders used to determine fragment lengths.  The short bands near the bottom are produced by the primers interacting among themselves during PCR.
 
 

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