Oligo primers

For all real-time PCRs, it is a good idea to verify that all of the oligos (primers and probe) that will be used together in the same reaction will not form dimers, particularly at the 3 ends. The 3 complementarity can be checked by scanning the sequences manually. If you are using primer design software, the program itself may mn a check to make sure that the sequence choices it picks are not complementary to each other. 

DNA/RNA hybrid primer, instead of all RNA oligo primer, is used here for more stable and economical purposes. All RNA primer would cost more to make and less stable. Also, due to DNA nucleotides are on the 5 -end, ligation of the hybrid primer to 3 -end of RNA (or primer self-ligation) is avoided by RNA ligase. 

Differential display is a method for identifying differentially expressed genes, using anchored oligo-dT, random oligonucleotide primers and polymerase chain reaction on reverse-transcribed RNA from different cell populations. The amplified complementary DNAs are displayed and comparisons are drawn between the different cell populations. 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

RNA to initiate cDNA synthesis. All cellular mRNA contains multiple repeats of adenine bases (poly-A tails). Therefore the complementary thymine bases (oligo-dT) can be used as a primer that binds to the mRNA template required for the reverse transcriptase to synthesize the cDNA. In the case of pancreatic mRNAs (Figure 4.2), the signihcantly higher mRNA for insulin compared with other proteins allowed success in isolating the insulin-specihc cDNA. Subsequent insertion of cDNA into a bacterial expression vector allowed the production of functional insulin that is now marketed as a successful therapeutic product (Figure 4.2). 

A third alternative starts with an extract of RNA, not DNA. Mature eukaryotic mRNA contains a long run or tail of adenine residues at its 3 end. The poly(rA) tail can be hybridized with an oligomer of thymine residues, and the oligo(dT) can then be used as a primer for a particular kind of DNA polymerase known as reverse transcriptase. This enzyme, a polymerase associated with retroviruses, will use RNA as a template to make a complementary DNA copy of the RNA, creating a DNA-RNA double-stranded hybrid. In another round of synthesis, the enzyme can replace the RNA strand entirely with DNA, so that the RNA-DNA hybrid is completely converted to double-stranded DNA containing an exact copy of the original RNA sequence. This DNA molecule is known as cDNA because it has a strand that is complementary to (or a copy of) the original RNA. 

Fig. 8.2.2 Principle of genomic quantification by multiplex ligation-dependent probe amplification (MLPA). Synthetic oligonuleotides are designed to bind to exon 1 (oligo 1a and 1b) and 2 (2a and 2b). After specific hybridisation, the oligos are ligated. The ligation products undergo quantitative polymerase chain reaction using universal primers X and Y...

ImENA template is annealed to synthetic oligonucleotide (oligo dT) primer. 

Synthesis of cDNA, usually in radiolabeled form is accomplished with reverse transcriptase, the enzyme from retroviruses that synthesize a DNA-RNA hybrid from ssRNA.570 572 A short oligo (dT) primer is usually hybridized to the 3 poly (A) tail to initiate synthesis. Reverse transcriptase also has ribonuclease (RNase H) activity and will digest away the RNA. If desired, synthesis of the second strand can be carried out by a DNA polymerase to give a complete DNA duplex. Many gene sequences have been deduced from cDNA copies. 

Primers can be custom synthesized commercially at a fairly reasonable rate. Primer sequences are selected so that they are typically 21 to 24 nucleotides in length with an average G + C content of 40 to 60%. The primers should not be part of repetitive-sequence DNA, nor should the DNA have palindromic sequences. There are computer programs to help select oligonucleotide primers for PCR, such as PRIMER (from White Head Institute www-genome. wi. mit. echi/ftp //distribution/software/primer. 0.5/manual. asc). In addition, commercial sources exist for software (National Biosciences) that includes the OLIGO program. 

Braun A, Little DP, Koster H. Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry. Clin Chem 1997 43 1151-1158. 

OLIGO start LEFT PRIMER 632 RIGHT PRIMER 840 SEQUENCE SIZE 1071... 

---