Introduction
The polymerase chain reaction (PCR) is an incredibly versatile technique that has made a profound impact on many areas of biology. PCR is based on the use of oligonucleotides, which flank the region of DNA of interest and which are complementary to sequences on either DNA strand, to prime the replication of each strand by Taq DNA polymerase. This enzyme is thermostable, and consequently, one can go through cycles of template denaturation and rena-turation after each round of replication and exponentionally amplify the sequence of interest. This procedure is very sensitive, and as such, it allows one to amplify, isolate, and utilize specific DNA sequences from vanishingly small quantities of starting material using either genomic or cDNA as a substrate.
PCR can also be used to facilitate the isolation of genes that are evolution-arily related, such as cognate genes from different species or multiple members of given gene families. In these cases, one is often trying to isolate DNA fragments whose sequence is not actually known. This can be done by taking advantage of the fact that the conservation of molecules is most evident at the protein level. In these cases, the sequence of the primers to be used is derived from conserved amino acid sequences that exist between the related genes. Since usually more than one codon encodes most amino acids (Fig. 1), and also since the amino acid sequence conservation between proteins may not be 100%, if the primers are to cover all of the possible combinations of DNA sequence that could encode a given amino acid motif, they will necessarily be degenerate. This chapter will focus on the use of degenerate oligonucleotide primers in PCR on cDNA synthesized from embryonic tissue. Using cDNA as
From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ
Amino acid
Alanine
Cysteine
Aspartic acid
Glutamic acid
Phenylalanine
Glycine
Histidine
Isoleucine
Lysine
Leucine
Methionine
Asparagine
Proline
Glutamine
Arginine
Serine
Threonine
Valine
Tryptophan
Tyrosine
Ala A Cys C Asp D Glu E Phe F Gly G His H Isl I Lys K Leu L
Codons
GCA GCC GCG GCU
UGC UGU GAC GAU GAA GAG
UUC UUU GGA GGC GGG GGU CAC CAU AUA AUC AUU AAA AAG
UUA UUG CUA CUC CUG AUG
AAC AAU
CCA CCC CCG CCU CAA CAG
AGA AGG CGA CGC CGG CGU AGC AGU UCA UCC UCG
ACA ACC ACG ACU GUA GUC GUG GUU UGG
UAC UAU
Fig. 1. The genetic code. Each of the amino acids is shown along with its corresponding RNA codons.
a substrate with degenerate oligonucleotides rather than genomic DNA has the advantage that one does not need to worry about introns and also that if the appropriate fragment is amplified from a cDNA sample, one can therefore conclude that this gene is expressed in the tissue from which the RNA was isolated. This allows one to amplify cDNA fragments of genes of interest that have not already been isolated from that organism, and also to survey what members of a given gene family, be they previously identified or not, are expressed in a given tissue at any particular stage of interest and to isolate cDNA fragments for each gene.
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