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Project Documentation & Protocols: Maize Gene Discovery Project: Education: Contents: Maize Gene Discovery | The Challenge of Maize Genetics | Why Discover Maize Genes? | Finding Genes Complementary DNA (cDNA), is produced in the laboratory by extracting an organism's single-stranded messenger RNA and transcribing it back into a stable piece of double-stranded DNA. The cDNA sequence does not include portions of the genomic DNA (introns) that were spliced out of the mRNA while it was still in the cell. Researchers can make multiple copies of cDNAs from a particular tissue and clone them into plasmids for storage and future study. cDNAs can be sequenced to identify ESTs and are also plated onto microarrays. Both techniques help identify which genes are turned "on" in a particular tissue.
ESTs, or expressed sequence tags, are pieces of the genetic sequence for genes that are turned "on," i.e., actively being transcribed into messenger RNA in the cell. To find a corn EST, researchers perform the following steps:
Gene, DNA, mRNA: Defining these terms is beyond the purpose of this website. For a general overview of DNA, see: http://www.dnaftb.org/. For a general discussion of molecular genetics, see: Molecular Genetics: Piecing it Together: http://www.ncbi.nih.gov/About/primer/genetics_molecular.html What is a Genome: http://www.ncbi.nih.gov/About/primer/genetics_genome.html Genome: A genome is the complete nucleotide sequence of an organism's DNA. Microarrays are plates or slides dotted with a grid of individual cDNAs. When mRNA extracted from a tissue is tagged with a fluorescent dye and poured over the microarray, it binds to its matching cDNA and lights up that dot. This allows scientists to study gene expression . i.e., which genes are turned "on" in a particular tissue at a particular time and under specific conditions. Non-coding DNA: Some proportion of chromosomal DNA consists of genes -- regions that code for proteins. But the rest is non-coding DNA: It may or may not serve a purpose . such as controlling which genes are active -- but it does not get transcribed into mRNA in order to make cellular proteins. PCR or Polymerase Chain Reaction: A procedure for cloning many copies of a gene of interest. A researcher looking for a particular gene can use PCR. First, she creates small, radioactively or flourescently-labelled pieces of DNA with a sequence that matches part of the gene she seeks. She adds these "primers" to a mixture of single-stranded DNA from an organism. The primers seek out their complementary base pairs in the organismal DNA and bind to them. The researcher then adds all the necessary ingredients to make the primer extend itself along the organismal DNA, growing a clone of the desired genetic material. By cycling the mixture through various temperatures, many clones are produced. The technique's inventor, Kary Mullis, won a Nobel Prize for developing this powerful procedure, now widely used for genetic and forensic testing. Plasmids: Pieces of bacterial DNA that are not part of the bacterial chromosome. They exist naturally inside many bacteria but have also become important tools of molecular biology. Plasmids often carry antibiotic resistance genes. When bacteria are grown on plates containing antibiotics, those that carry such plasmids will survive. In this way, researchers can select and study colonies that contain DNA of interest. Plasmid Libraries: To create DNA samples that can be studied well into the future, researchers insert the DNA into a plasmid and then place the plasmid inside E. Coli where it can be saved on frozen plates. These plates are called plasmid libraries. Recessive vs. Dominant alleles: Organisms with two copies of each chromosome typically carry at least two copies or "alleles" of each gene. These alleles often differ from one another, and one may dominate over the other. For example, a person who has one allele for brown eyes and one for blue will have brown eyes because the brown-eyed allele is dominant while the blue-eyed allele is recessive. RescueMu is a transposon that was genetically engineered at Stanford. Unlike many transposons, RescueMu does not jump from place to place as the plant grows. Instead, it copies itself and inserts into maize genes primarily during meiosis . the formation of eggs and pollen. After inserting, it does not move. This type of transposon movement is called replicative transposition rather than "cut and paste." The RescueMu transposon was designed with a known sequence of nucleotide repeats at each end. It also includes a gene for antibiotic resistance. These features help researchers find the transposon after it has been copied into a new location. Retrotransposon is a name for mobile transposon elements which use RNA as a template for replication. Splicing: After DNA is transcribed into mRNA in the nucleus, the mRNA is edited: introns are spliced out and the remaining exons are joined together. In some instances, the editorial process produces several alternative versions of the mRNA, which in turn generates various proteins when it is transcribed. Transposon: A transposon is a mobile segment of DNA that serves as an agent of genetic change. Some transposons move by being cut and pasted into new locations. Others stay put but insert copies of themselves elsewhere in the genome, a process called replicative transposition. The two ends of a transposon carry a sequence of bases that permit the transposase enzyme to clip and move or copy the DNA segment to new locations in a cell's DNA. For More Life Science and Genetics Glossaries, check out:
Katherine Miller, a freelance science writer, contributed the text for this page to the Maize Gene Discovery Project. You can reach her at [email protected]. Return to Documentation Index | Return to Maize Gene Discovery Project index | Return to Homepage |
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