By Melissa Rivera

 GENE CLONING: production of multiple identical copies of DNA  It was developed so scientists could work directly with specific genes  Cloned genes purposes: a. make many copies of particular gene b. produce protein product  Restriction Enzymes: cut DNA molecule into specific nucleotide sequences to yield a set of double-stranded DNA fragments with single-stranded sticky ends.  Sticky ends form hydrogen bonded base pairs with complementary sticky ends on any other DNA molecule cut with the same enzyme.  DNA ligase seals the base-paired fragments producing recombinant DNA molecules.  Cloning vector is the original plasmid that can carry foreign DNA into a cell and replicate there.  Once a cell is cloned it can be identified with a radioactively labeled nucleic acid probe which has a sequence complementary to the gene  Genomic Library: collection of recombinant vector clones produced by cloning DNA fragments derived from an entire genome  Complementary DNA: a DNA molecule made in vitro using mRNA as a template and the enzyme reverse transcriptase  cDNA Library: limited gene library using cDNA; it includes only genes that were transcribed in the cells examined  Biologists use yeast cells to avoid eukaryotic/prokaryotic incompatibility. Using cultured eukaryotic cells as host cells and yeast artificial chromosomes as vectors helps avoid those problems.  Electroporation: a technique to introduce recombinant DNA into cells by applying brief electrical pulse to a solution containing cells  Polymerase Chain reaction (PCR): a technique in which any specific target segment within one or many DNA molecules can be quickly amplified or copied many times in a test tube.

 Gel Electrophoresis: technique that uses a gel as a molecular sieve to separate nucleic acids or proteins on the basis of size, electrical charge and other physical properties. Not only are the restriction fragments sorted but gel electrophoresis provides a way to prepare pure samples of individual fragments., and to compare 2 different DNA molecules.  Southern Blotting: a hybridization technique that enables researchers to determine the presence of certain nucleotide sequences in a sample of DNA.  Restriction Fragment Length Polymorphisms : differences in DNA sequence on homologous chromosomes that result in restriction fragments of different lengths, that can be detected by southern blotting.  RFLPS enabled geneticists to not be limited by genetic variations that lead to phenotypic differences or differences in protein products.

 DNA variations reflected in RFLPs serve as a basis for an extremely detailed map of the entire human genome. Human Genome Project : the entire nucleotide sequence of the majority of DNA in each human chromosome was obtained. Three Stages: a. genetic or linkage mapping b. physical mapping c. DNA sequencing  Linkage Map: a genetic map based on the frequencies of recombination between markers during crossing over of homologous chromosomes.  Physical Map: the distances between markers are expressed in some physical measure; usually the number of base pairs along the DNA. It is constructed by cutting a DNA molecule into many short fragments and arranging them in order by identifying overlaps.  Dideoxyribnucleotide chain determination method: a sequencing technique developed by Frederick Sanger., which can be performed in automated sequencing machines.

 Genomics: study of whole sets of genes and their interactions.  Expressed Sequence tags are sequences cataloged in computer databases that can be analyzed to identify sequences that may be new protein coding genes, so called putative genes or gene candidates.  Genome size doesn’t correlate to the complexity of the organism.  2 ways to determine the function of new genes: a. In vitro mutagenesis: specific mutations are introduced into the sequence of a cloned gene b. RNA interference: uses synthetic double stranded RNA molecules matching the sequence of a particular gene to trigger breakdown or to block translation of the gene’s mRNA.  DNA Microarray assay: method to detect and measure the expression of thousands of genes at one time.  Comparisons of genome sequences from different species allow us to determine evolutionary relatioships. These comaprative studies provide valuable information in biology.  Proteomics: systematic study of all the proteins encoded by a genome  SNPs: provide useful markers for studying human genetic variation

 Medical Applications: by identifying human genes whose mutation plays a role in genetic diseases, it can lead to ways of diagnosing, treating, and preventing such conditions. Gene Therapy: the alteration of an inflicted individual’s genes. It holds great potential for treating disorders traceable to a single defective gene.  Pharmaceutical Products: DNA technology can be used to develop vaccines that stimulate the immune system to defend against specific pathogens.  Forensic evidence: DNA testing can identify the guilty individual because the DNA sequence of every person is unique. DNA fingerprint or specific pattern bands can be tested to identify the guilty person. Fingerprinting can also be used to establish paternity. Fingerprinting is reliable because the probability of having identical finger prints is extremely rare.  Environmental Cleanup: Genetic engineering can be used to modify metabolism of microorganisms so that they can be used to extract minerals from the environment or degrade various types of toxic waste materials.  Agriculture: DNA technology is being used in an effort to improve productivity. Transgenic: scientist introduce a gene from one animal into the genome of another animal. The goal is to improve productivity and food quality.  Safety and Ethical questions: Today a major concern is the use of genetically modified organisms (one acquired by artificial means) as food.