Chapter 12

Genetic Technologies  Biotechnology  manipulation of organisms or their components to make useful products  Wine, cheese  Livestock selective breeding  DNA technology  techniques used to study and manipulate genetic material

Genetic Engineering  manipulating genes for practical purposes  Gene cloning  leads to the production of multiple, identical copies of a gene-carrying piece of DNA  Recombinant DNA  formed by joining nucleotide sequences from two different sources  One source contains the gene that will be cloned  Another source is a gene carrier  vector  Plasmids often used

Genetic Engineering  Restriction enzymes  cut DNA at specific sequences  Each enzyme binds to DNA at a different restriction site  Many restriction enzymes make staggered cuts that produce restriction fragments with single- stranded ends  “sticky ends”  Fragments with complementary sticky ends can associate with each other, forming recombinant DNA  DNA ligase joins DNA fragments together

A restriction enzyme cuts the DNA into fragments. Restriction enzyme recognition sequence Restriction enzyme Gene of interest A DNA fragment from another source is added. Two (or more) fragments stick together by base pairing. Sticky end Sticky end DNA ligase pastes the strands together. Recombinant DNA molecule DNA

1.Plasmid DNA isolated 2.DNA containing the gene of interest is isolated 3.Plasmid DNA is treated with a restriction enzyme 4.DNA with the target gene is treated with the same enzyme and many fragments are produced 5.Plasmid and target DNA are mixed and associate with each other 6.Recombinant DNA molecules produced when DNA ligase joins plasmid and target segments together 7.Recombinant plasmid containing the target gene taken up by a bacterial cell Then binary fission!! Steps in Cloning a Gene

E. coli bacterium Bacterial chromosome A plasmid is isolated. Gene of interest The plasmid is cut with an enzyme. Plasmid The cell’s DNA is isolated. The cell’s DNA is cut with the same enzyme. DNA Examples of gene use A cell with DNA containing the gene of interest Gene of interest The targeted fragment and plasmid DNA are combined. DNA ligase is added, which joins the two DNA molecules. Gene of interest Genes may be inserted into other organisms. The recombinant plasmid is taken up by a bacterium through transformation. Examples of protein use Harvested proteins may be used directly. The bacterium reproduces. Clone of cells Recombinant bacterium Recombinant DNA plasmid

Recombinant cells and organisms  used to manufacture many useful products, chiefly proteins  Bacteria often used because:  have plasmids and phages available for use as gene- cloning vectors  can be grown rapidly and cheaply  can be engineered to produce large amounts of a particular protein  often secrete the proteins directly into their growth medium

 human insulin gene isolated and cut from its location on the human chromosome ◦ using a restriction enzyme  plasmid is cut using the same restriction enzyme  desired DNA (insulin gene) and plasmid DNA can be joined using DNA ligase  plasmid now contains the genetic instructions on how to produce the protein insulin  Bacteria can be artificially induced to take up the recombinant DNA plasmids and be transformed ◦ successfully transformed bacteria will contain the desired insulin gene  transformed bacteria containing the insulin gene can be isolated and grown  As transformed bacteria grow they will produce the insulin proteins coded for the recombinant DNA ◦ Insulin harvested and used to treat diabetes

Genetically modified organisms  contain one or more genes introduced by artificial means  GM plants are being produced that  are more resistant to herbicides and pests  provide nutrients that help address malnutrition  GM animals are being produced with improved nutritional or other qualities  Are they safe?!?!

DNA PROFILING

DNA Profiling  analysis of DNA fragments to determine whether they come from the same individual  compares genetic markers from noncoding regions that show variation between individuals  involves amplifying (copying) of markers for analysis

DNA is isolated The DNA of selected markers is amplified. The amplified DNA is compared. Crime sceneSuspect 1Suspect 2

PCR  Polymerase chain reaction (PCR)  method of amplifying a specific segment of a DNA molecule  PCR  three-step cycle  doubles the amount of DNA in each turn of the cycle  Advantages: ◦ amplify DNA from a small sample ◦ obtaining results rapidly ◦ highly sensitive, copying only the target sequence

Gel electrophoresis  used to separate DNA molecules based on size 1.A DNA sample is placed at one end of a gel 2.Current is applied and DNA molecules move from the negative electrode toward the positive electrode 3.Shorter DNA fragments move through the gel matrix more quickly and travel farther through the gel 4.DNA fragments appear as bands, visualized through staining 5.Each band is a collection of DNA molecules of the same length

A mixture of DNA fragments of different sizes Power source Gel Completed gel Longer (slower) molecules Shorter (faster) molecules

 In October 2001, Florida man died from inhalation anthrax ◦ By the end of the year, four other people had also died from anthrax  Investigators analyzed the genome of the anthrax spores used in each attack ◦ Able to establish that the spores from all of the cases were identical  Suggested a single perpetrator of the crime  Able to match the anthrax with one laboratory subtype  The Ames strain

 Betty Anne Waters ◦ Ayer, MA  1982 – brother arrested for murder  Waters went to CCRI ◦ GED ◦ Associates ◦ Went to Roger Williams to get Bachelors and Law degree  Became brothers lawyer ◦ Witnesses lied**** ◦ DNA evidence in 1990’s ◦ Innocence Project  Released in 2001 after serving 18 years in prison ◦ $3.4 million dollar settlement /

GENOMICS

Genomics  study of an organism’s complete set of genes and their interactions  Initial studies focused on prokaryotic genomes  Many eukaryotic genomes have since been investigated

Genomics  allows another way to examine evolutionary relationships  showed a 96% similarity in DNA sequences between chimpanzees and humans  Functions of human disease-causing genes have been determined by comparing human genes to similar genes in yeast

The Human Genome Project  determining the nucleotide sequence of all DNA in the human genome  identifying the location and sequence of every human gene  revealed that most of the human genome does not consist of genes

The Human Genome Project  Results indicate that:  humans have about 20,000 genes in 3.2 billion nucleotide pairs  only 1.5% of the DNA codes for proteins, tRNAs, or rRNAs  remaining 98.5% of the DNA is noncoding DNA  Telomeres  stretches of noncoding DNA at the ends of chromosomes  transposable elements,  DNA segments that can move or be copied from one location to another within or between chromosomes

Proteomics  Proteomics  study of the full protein sets encoded by genomes  investigates protein functions and interactions  human proteome includes about 100,000 proteins

Genomes hold clues to human evolution  Human and chimp genomes differ by:  1.2% in single-base substitutions  2.7% in insertions and deletions of larger DNA sequences  Genes showing rapid evolution in humans include  genes for defense against malaria and tuberculosis  a gene regulating brain size  the FOXP2 gene involved with speech and vocalization