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The role of probe-probe interactions on the hybridization of double-stranded DNA targets onto DNA-modified magnetic microparticles.

IBB-Institute for Biotechnology and Bioengineering, Center for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.

In this work, we have studied the effect of different probe lengths and surface densities on the hybridization of a 181-bp polymerase chain reaction product to probes tethered onto magnetic microparticles. Hybridization was shown to be favored by longer probes but only at probe surface densities where probe-to-probe interactions are absent. From these results, a simple rule was inferred for determining maximum surface densities above which hybridization signals decreased. According to this rule, if the average surface area occupied by an immobilized probe (Sigma) is larger than the projected surface area of each tethered probe molecule (S ( ss )), hybridization efficiency increases with surface density, whereas the reverse occurs when Sigma - S ( ss ) < 0.

PMID: 19462163 [PubMed - as supplied by publisher]

The cut is made between the adjacent G and C. This particular sequence occurs at 11 places in the circular DNA molecule of the virus phiX174. Thus treatment of this DNA with the enzyme produces 11 fragments, each with a precise length and nucleotide sequence. These fragments can be separated from one another and the sequence of each determined.

Restriction Enzymes

Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases.Restriction enzymes can also differ in the way they cut the DNA molecule. Some enzymes cut in the middle of the recognition sequence, resulting in a flush or blunt end. Other enzymes cleave in a staggered fashion, resulting in DNA products that have short single-stranded overhangs (usually two or four nucleotides) at each end. These are often called cohesive ends, as these single-stranded overhangs could potentially come together again through complementary base-pairing

 A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIII that cuts DNA wherever it encounters the sequence In this laboratory you will treat bacteriophage lambda DNA with either the restriction enzyme Hind III or Eco RI. This restriction enzyme digest will create a pattern of specific DNA fragments that serves as a "DNA ladder. " Since the entire DNA sequence of the bacteriophage is known, we know exactly where a restriction enzyme will cleave the viral DNA and the size of the DNA fragments generated.