Biotinylated Primers [was SS PCR]

[Ernest Retzel 1535 49118]

>How about a reference or two on that.  I am not sure about how to biotinylate 
>the primer, ie how many biotins per primer, how much does the biotin affect the 
>PCR reaction , and how do you determine how many/much of the magnetic particles 
>do you add per estimated biotin content?

Generally, I put the biotin directly on during synthesis on the 5'-terminus.
Cyanoethyl phosphoramidite biotin can be bought from several sources; my
personal favorite is Glen Research, Inc., in Sterling, VA [800-327-GLEN; fax:
703-435-9774] [yes, all usual disclaimers].  Their only business is nucleic
acid synthesis reagents, and they do it well.

I am not where my paper files are [as in, I am at home...], but a quick
search of MedLine with keywords "biotin*" and "magnetic" came up with the
following [and a few more]; there were others with 'pcr' and 'polymerase chain
reaction,' coupled with these terms, but I didn't have the patience to sort
them at 9600 baud...  The magnetic bead folks [Dynex?] have an extensive
literature file of their own as well.  Their number I don't have here.  With
all the usual disclaimers, they are pretty much the clear favorite, even though
they are not the cheapest.

Ernest Retzel
Dept. of Microbiology
University of Minnesota

ernest at lenti.med.umn.edu

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AU  - Kaneoka H;Lee DR;Hsu KC;Sharp GC;Hoffman RW;
TI  - Solid-phase direct DNA sequencing of allele-specific polymerase chain
      reaction-amplified HLA-DR genes.
SO  - Biotechniques 1991 Jan;10(1):30, 32, 34
AB  - We describe in this report a new strategy to directly sequence
      polymerase chain reaction-amplified human leucocyte antigen DRB genes
      using biotinylated allele-specific synthetic oligonucleotide primers
      coupled to streptavidin-coated magnetic beads. The use of
      allele-specific primers in the polymerase chain reaction allows for
      selective amplification of DNA from one haplotype, which when combined
      with the direct sequencing technique circumvents the need for DNA
      cloning prior to sequencing. We demonstrate here that this method can be
      used to characterize human leucocyte antigen DRB genes among
      heterozygous individuals. This method can be used for the rapid analysis
      of highly polymorphic genes among individuals heterozygous at the gene
      of interest.

AU  - Shaffer AL;Wojnar W;Nelson W;
TI  - Amplification, detection, and automated sequencing of gibbon
      interleukin-2 mRNA by Thermus aquaticus DNA polymerase reverse
      transcription and polymerase chain reaction.
SO  - Anal Biochem 1990 Nov 1;190(2):292-6
      ANALYTICAL BIOCHEMISTRY (NEW YORK)
AB  - Reverse transcription-polymerase chain reaction (RT-PCR) is a gene
      expression assay by which messenger RNA (mRNA) production can be
      measured. This technique involves three steps: isolation of RNA from
      cells or tissues, the creation of a DNA copy of the desired message
      (cDNA) by viral reverse transcriptase enzymes (RT), and amplification of
      this DNA segment by the polymerase chain reaction (PCR) for subsequent
      quantitation and analysis. Here we describe a one-enzyme, one-step
      method combining the RT and PCR steps of conventional RT-PCR by
      exploiting the recently documented RT properties of Taq polymerase, the
      thermostable enzyme used for PCR amplification of DNA. RNA was extracted
      from gibbon T-cells (MLA144), reverse transcribed and amplified with
      oligonucleotide primers (specific for the 5' portion of a spliced
      interleukin-2 (IL-2) messenger RNA) by Taq polymerase. A discrete
      fragment of correct length for IL-2 cDNA was detected. Experiments
      showed that this product was RNA-dependent and specific for IL-2. This
      fragment was sequenced by automation employing a biotin
      primer-streptavidin magnetic bead protocol which confirmed its origin as
      processed IL-2 mRNA. The modification of the RT-PCR procedure using a
      thermostable enzyme speeds up reaction time and increases stringency.
      This method should make the diagnostic screening of cells for gene
      expression more efficient and practical.

AU  - Rimstad E;Hornes E;Olsvik O;Hyllseth B;
TI  - Identification of a double-stranded RNA virus by using polymerase chain
      reaction and magnetic separation of the synthesized DNA segments.
SO  - J Clin Microbiol 1990 Oct;28(10):2275-8
      JOURNAL OF CLINICAL MICROBIOLOGY (WASHINGTON)
AB  - A double-nested polymerase chain reaction assay (PCR), followed by
      magnetic separation of the PCR-synthesized DNA segments, was developed
      to detect a double-stranded RNA virus, infectious pancreatic necrosis
      virus from salmonid fish. Viral RNA was extracted from cell cultures and
      used for cDNA synthesis. The cDNA produced was used as a template in a
      double PCR. The sensitivity of this double PCR was approximately 0.8 pg
      of template double-stranded RNA. The DNA segment produced from the first
      PCR was also used as a template in a second PCR with a set of two
      5'-labeled primers, one with biotin and the other with 32P. The PCR
      segment that was then synthesized was separated from the solution by
      using streptavidin-coated, superparamagnetic beads. The levels of
      radioactivity measured in the magnetically separated fractions were
      significantly higher in the positive samples than they were in the
      negative samples.

AU  - Wahlberg J;Lundeberg J;Hultman T;Holmberg M;Uhlen M;
TI  - Rapid detection and sequencing of specific in vitro amplified DNA
      sequences using solid phase methods.
SO  - Mol Cell Probes 1990 Aug;4(4):285-97
      MOLECULAR AND CELLULAR PROBES (LONDON)
AB  - We describe a rapid solid phase assay for detection and sequencing of
      DNA sequences based on selective introduction of biotin and isotope into
      the specific DNA fragment amplified by the polymerase chain reaction
      (PCR). A two-step PCR procedure is used to lower the background signal.
      The in vitro amplified material is immobilized on magnetic beads with
      covalently coupled streptavidin and the amount of bound label is
      measured. Samples identified as positive can be analysed by direct solid
      phase DNA sequencing. A strategy is also described to use general
      primers for detection, capturing and sequencing, which are not
      homologous to the specific sequence to be detected. The concept has been
      optimized using oligonucleotides specific for Staphylococci and
      Streptococci, respectively. Here, we show that the assay can be used for
      detection of Plasmodium falciparum in clinical samples.


AU  - Hunsaker WR;Badri H;Lombardo M;Collins ML;
TI  - Nucleic acid hybridization assays employing dA-tailed capture probes. II.
      Advanced multiple capture methods.
SO  - Anal Biochem 1989 Sep;181(2):360-70
      ANALYTICAL BIOCHEMISTRY (NEW YORK)
AB  - A fourth capture is added to the reversible target capture procedure of
      the preceding paper. This results in an improved radioisotopic detection
      limit of 7.3 x 10(-21) mol of target. In addition, the standard triple
      capture method is converted into a nonradioactive format with a
      detection limit of under 1 amol of target. The principal advantage of
      nonradioactive detection is that the entire assay can be performed in
      about 1 h. Nucleic acids are released from cells in the presence of the
      ('capture probe') which contains a 3'-poly(dA) sequence and the
      ('labeled probe') which contains a detectable nonradioactive moiety such
      as biotin. After a brief hybridization in solution, the target is
      captured on oligo(dT) magnetic particles. The target is further purified
      from sample impurities and excess labeled probe by recapture either once
      or twice more on fresh magnetic particles. The highly purified target is
      then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose
      filter and rapidly detected with streptavidin-alkaline phosphatase using
      bromochloroindolyl phosphate and nitroblue tetrazolium. Using this
      procedure, as little as 0.25 amol of a target plasmid has been detected
      nonradioactively in crude samples in just 1 h without prior purification
      of the DNA and RNA. Finally, a new procedure called background capture
      is introduced to complement the background-reducing power of RTC.

AU  - Hultman T;St ahl S;Hornes E;Uhlen M;
TI  - Direct solid phase sequencing of genomic and plasmid DNA using magnetic
      beads as solid support.
SO  - Nucleic Acids Res 1989 Jul 11;17(13):4937-46
      NUCLEIC ACIDS RESEARCH (LONDON)
AB  - Approaches to direct solid phase sequencing of genomic and plasmid DNA
      have been developed using magnetic beads, coated with streptavidin, as
      solid support. The DNA is immobilized through selective incorporation of
      biotin into one of the strands. A single stranded template, suitable for
      sequencing, is obtained through strand-specific elution. Using this
      concept, in vitro amplified plasmid DNA and chromosomal DNA were
      sequenced directly from single colonies. The solid phase approach
      ensures that the amplification and the sequencing reactions can be
      performed under optimal conditions. The system was found to be suitable
      for sequencing using both isotope- and fluorescent-labelled primers.

AU  - Dudin G;Steegmayer EW;Vogt P;Schnitzer H;Diaz E;Howell KE;Cremer T;
      Cremer C;
TI  - Sorting of chromosomes by magnetic separation.
SO  - Hum Genet 1988 Oct;80(2):111-6
      HUMAN GENETICS (BERLIN)
AB  - Chromosomes were isolated from Chinese hamster x human hybrid cell lines
      containing four and nine human chromosomes. Human genomic DNA was
      biotinylated by nick translation and used to label the human chromosomes
      by in situ hybridization in suspension. Streptavidin was covalently
      coupled to the surface of magnetic beads and these were incubated with
      the hybridized chromosomes. The human chromosomes were bound to the
      magnetic beads through the strong biotin-streptavidin complex and then
      rapidly separated from nonlabeled Chinese hamster chromosomes by a
      simple permanent magnet. The hybridization was visualized by additional
      binding of avidin-FITC (fluorescein) to the unoccupied biotinylated
      human DNA bound to the human chromosomes. After magnetic separation, up
      to 98% of the individual chromosomes attached to magnetic beads were
      classified as human chromosomes by fluorescence microscopy.