Biogenetic Engineering & Manipulating Genes Chapter 20

Intro. Q’s #5 for Chapter 20: Genetic Engineering 1)What does the acronym PCR stand for and what does this process do? 2)What does Gel electrophoresis allow us to do? 3)Give two applications of DNA profiling. 4)What are the advantages and disadvantages of genetic screening? 5)How is a cDNA library different from a genomic library? (p ) 6)Name two “vectors” that can be used for gene transfer. 7)Give two examples of a genetically modified crop or animal. 8)Briefly explain the process of gene therapy and give an example how it works. 9)Explain what a clone is and how it could be formed. 10)What are some of the ethical concerns about cloning? Give your opinion if you think cloning is something we should be doing.

Genetic Engineering Chapter 20 DNA Technology & Genomics Pgs &

O.J. Simpson capital murder case,1/95-9/95 Odds of blood in Ford Bronco not being R. Goldman’s: 6.5 billion to 1 Odds of blood on socks in bedroom not being N. Brown-Simpson’s: 8.5 billion to 1 Odds of blood on glove not being from R. Goldman, N. Brown-Simpson, and O.J. Simpson: 21.5 billion to 1 Number of people on planet earth: 6.1 billion Odds of being struck by lightning in the U.S.: 2.8 million to 1 Odds of winning the Illinois Big Game lottery: 76 million to 1 Odds of getting killed driving to the gas station to buy a lottery ticket 4.5 million to 1 Odds of seeing 3 albino deer at the same time: 85 million to 1 Odds of having quintuplets: 85 million to 1 Odds of being struck by a meteorite: 10 trillion to 1

Introduction Manipulating genes and sequencing DNA has become one of the greatest achievements in the last 50 years -First complete genome sequenced was a bacteria (1995) -Human Genome project ( )

Recombinant DNA Def: DNA in which genes from 2 different sources are linked Genetic engineering: direct manipulation of genes for practical purposes Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

Tools for DNA Analysis & Genomics PCR (polymerase chain reaction) Gel electrophoresis Restriction fragment analysis (RFLPs) Southern blotting DNA sequencing Human genome project

Tools of Genetic Engineering Restriction enzymes (endonucleases) -in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific Restriction site: -recognition sequence for a particular restriction enzyme Restriction fragments: -segments of DNA cut by restriction enzymes in a reproducable way Sticky end: -short extensions of restriction fragments DNA ligase: -enzyme that can join the sticky ends of DNA fragments Cloning vector: -DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids)

Restriction Enzymes (endonucleases)

Producing Restriction Fragments DNA ligase used to splice together cut plasmids and chromosome fragments

Cloning

Steps for Eukaryotic Gene Cloning (pg. 387) Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest) Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase Introduction of cloning vector into cells (transformation by bacterial cells) Cloning of cells (and foreign genes) Identification of cell clones carrying the gene of interest

DNA Libraries (pg. 388) Collection of DNA fragments that have been incorporated into plasmids

Genomic Libraries (pg. 388) Stored amount of cloned genes within bacteria or virues (bacteriophages) Contained as cloned plasmids within bacteria or as fragments within Bacteriophages genome Phages can carry a larger DNA insert vs. bacteria

cDNA Library (Pg. 390) cDNA (complementary DNA) Start w/mRNA to make a ssDNA strand Use an enzyme “reverse transcriptase” which is found in retroviruses such as HIV (see pg ) to make a ssDNA strand. A complementary strand of DNA is made by DNA polymerase to produce a dsDNA molecule (cDNA)

Polymerase Chain Reaction (PCR) Amplification of any piece of DNA without cells (in vitro) Produces many identical copies of a DNA segment Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers Applications: fossils, forensics, prenatal diagnosis, etc.

Polymerase Chain Reaction PCR = common method of creating copies of specific fragments of DNA –PCR rapidly amplifies a single DNA molecule into many billions of molecules. Small samples of DNA can produce sufficient copies to carry out forensic tests

Polymerase Chain reaction (PCR)

Inserting DNA into the nucleus of a cell

Gel Electrophoresis (Lab) DNA fragments placed into “wells” in gel agarose Electricity pulls on DNA fragments Fragments travel at different rates based on size and ability to squeeze through swiss-cheese-like agarose

Restriction Fragment Analysis Restriction fragment length polymorphisms (RFLPs) Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence DNA Fingerprinting (DNA Profiling)

Gel Electrophoresis separates nucleic acids or proteins on the basis of size and electrical charge creating DNA bands of the same length DNA has a net negative charge (use a positive charge in the gel)

Southern Blot of DNA Fragments (pg. 395) Used to determine the presence or absence of a particular nucleotide sequence in the DNA different sources. Compare the restriction fragments produced from the different samples. Radioactive probes are used and attach to the DNA fragments by complementary base pairing.

DNA Profiling (DNA fingerprinting) Applications: -Used in criminal investigations -Identify the remains of dead people -Compare Evolutionary origins of organisms -Look for genetic disorders (mutations) -Sequence DNA samples

DNA Fingerprinting

Video #3: Using DNA evidence to Solve Crimes Write 15 key statements from the video. Please number each statement. Indicate where the sources of DNA were found for each case.

DNA Sequencing Determination of nucleotide sequences (Sanger method, sequencing machine) Genomics: the study of genomes based on DNA sequences Human Genome Project

Practical DNA Technology Uses Diagnosis of disease Human gene therapy Pharmaceutical products (vaccines) Forensics Animal husbandry (transgenic organisms) Genetic engineering in plants Ethical concerns?

Genetic Screening Def: Testing individuals in a population for the presence or absence of a gene (allele) Advantages: -pre-natal diagnosis of genetic disorders -Could help stop the spread of a disorder -Can detect carriers of a potential disorder Disadvantages: -invasion of privacy -Individuals can become stigmatized in the community -Discriminated against or feared -Employment and medical insurance

Bacterial plasmids in gene cloning

Steps for Eukaryotic gene cloning

Plant Cloning Tissue Culture Propagation – Bits of phloem can be induced in the lab to form clumps of tissue that will make roots & shoots – Orchid culture

Embryo Cloning Medical technique which produces identical twins or triplets – Duplicates nature – One or more cells are removed from a fertilized embryo, encouraged to develop into one identical twins or triplets – Done for many years on animals – Limited experimentation on humans

Adult DNA Cloning Cell nuclear replacement –Produces a duplicate of an existing animal –DNA from an embryo is removed; –Replaced with DNA from an adult animal –Embryo is implanted in a womb and allowed to develop into a new animal Untried on humans - potential of producing a twin of an existing person

Therapeutic Cloning Stem cells removed from an embryo with intent of producing tissue or a whole organ for transplant back into the person who supplied the new DNA –Embryo dies in the process Goal is to produce a healthy copy of a sick person's tissue or organ for transplant

Application: Gene Therapy

Therapeutic Cloning Vastly superior to organ transplants – Supply would be unlimited - no waiting lists Tissue or organ would have the sick person's original DNA – No immunosuppressant drugs would need to be taken

Adult DNA Cloning

Stem cell clon- ing

Theraputic Stem Cell Cloning Used

Chromatin Def: complex of DNA and proteins DNA Packing Histone proteins (+ charged amino acids w/ phosphates of DNA that are - charged) Nucleosome -”beads on a string”; basic unit of DNA packing Heterochromatin -highly condensed interphase DNA (can not be transcribed) Euchromatin -less compacted interphase DNA (can be transcribed)