Chapter 12- DNA Technology and the Human Genome PCR Plasmid Probe R plasmid Recombinant DNA Restriction enzymes Restriction fragments Sex pili Sticky ends Telomeres Ti plasmid Transduction Transformation Transgenic organism Transposons Vaccine Vector Biotechnology Blunt ends cDNA Conjugation DNA fingerprinting DNA ligase DNA microarrays F factor Gel electrophoresis Gene cloning Gene therapy Genetic marker Genomic library Genomics GM organisms Golden rice Human Genome Project

Bacterial mating shows how genes are transferred Asexual reproduction 3 ways to transfer DNA: 1-Transformation- DNA is taken in from surrounding fluid Explains Griffiths experiment 2-Transduction- phage transfers bacterial genes Has genes from previous host

Conjugation- cells join and a copy of DNA is transferred via mating bridge Figures 12.1 A,B, and C in text pg 232

Plasmids in conjugation Small, circular piece of DNA separate from bacterial chromosome Carry genes during conjugation F-factor- piece of DNA that carries genes for sex pilli and other genes necessary for conjugation Behaves 2 ways during conjugation: 1- it is integrated into bacterial chromosome 2- it is a plasmid When it carries genes for more than replication and conjugation it is acting as a vector R –plasmids- “resistance” – carry genes for enzymes that destroy antibiotics They can also carry genes of interest and that gene can then be transcribed and translated

Plasmids in conjugation

What is biotechnology? Using organisms to perform practical tasks Bacteria making human proteins (insulin, HGH) Genes for pest resistance put into plants Bacteria with genes to break down toxic waste Golden Rice

How is this transformation done? Restriction enzymes- recognize short nucleotide sequences and cut at a specific point Used in nature to chop of intruder DNA to protect Cuts ends in two ways: sticky or blunt DNA ligase- catalyzes covalent bonds between nucleotides Putting genes into a piece of DNA using restriction enzymes and DNA ligase is producing recombinant DNA Genes can be cloned using this method

Gene Cloning Produces multiple copies of a gene Human DNA and bacterial plasmid are cut with same restriction enzyme They are mixed and with DNA ligase are recombined The plasmid is put back into the bacteria and allowed to reproduce asexually

Uses for recombinant DNA? Mass produce gene products “pharm” animals can produce human genes Human insulin can be produced by E. coli Certain proteins can now be produced and secreted in an animals milk Rice fortified with certain vitamins

GMO’s genetically modified organisms Organism that has artificially gained 1 or more genes To: delay ripening, resistance to spoiling, disease, herbicide, insecticide, add nutrients *Golden rice Vectors are usually used to introduce genes to plants (fig 12.18A) Ti plasmid- from soil bacteria (induces tumors) Researchers have removed tumor causing properties but kept ability to transfer DNA Recombinant organism is genes of another species is a transgenic organism

Keeping track of these pieces Genomic library- Set of DNA segments, carried on a plasmid or phage, from an organisms’ genome

Making cDNA (complementary) Using reverse transcriptase- produces DNA from RNA template DNA without introns can be transcribed and translated by bacteria Bacteria can then make human protein

Using probes Short labeled nucleic acid molecule Used to identify specific gene or DNA sequence Bacterial colonies are transferred to filter paper DNA is heated to separate strands Radioactive probes are added, filter is “developed” Compare “picture” with culture to find genes

DNA microarrays Used to detect the expression of 1000’s of genes at a time Tiny pieces of DNA are attached to glass and tested for hybridization with various DNA molecules

Gel Electrophoresis Sorts molecules by size and charge Samples are added to wells in a gel Electric current allows molecule movement Small molecules move farther and faster

Genetic Markers and probes are used with gel electrophoresis Genetic markers- chromosomal landmarks that can be used to study inheritance Non-coding sequences- similar in related individuals DNA is cut with restriction enzymes, and run through electrophoresis gel Probes can then be used to detect specific sequences

What if the sample of DNA we have it too small to run a gel? PCR- polymerase chain reaction amplifies amount of DNA in test tube without using living cells DNA sample is put into tube with DNA pol, nucleotides, and other molecules (primers) Sample is heated to separate strands then cooled for nucleotides to bind (done over and over again)

Uses for gel electrophoresis and PCR? DNA Fingerprinting- uses short repeats and PCR to amplify

Ironically… Most of your DNA is junk (97%)!!! DNA between genes is repetitive sequences 2 types: 1- short repeated sequences by centromere and at ends of chromosomes by telomeres- protect against DNA loss 2- 2nd type- long repeated sequences Scattered around genome “jumping genes”- transposons- can jump into middle of gene and disrupt expression

Barbara McClintock Worked at Cold Spring Harbor Lab Experimented with Indian corn kernel color and transposons

Uses in medicine: Therapeutic hormones- insulin, HGH PCR can help find viruses DNA microarrays- allows treatment to be tailored to a specific disorder Vaccines- harmless variant or derivative of a pathogen (usually bacteria or virus) Stimulates immune system to develop defenses against the pathogen 1 way- produce harmless artificial mutant of pathogen Causes fewer side effects 2nd way- engineer cells to produce protein that is found on pathogen’s surface

Gene Therapy Altering the afflicted individuals genes New allele can be inserted into somatic cells of infected tissue Bone marrow cells- good candidate because they include stem cells that can differentiate completely Bone marrow cells that have been infected with gene carrying virus are injected into afflicted individual New ideas focus on adding genes to help not fix Ex: heart growing new blood vessels There are ethical and technical problems

Human Genome Project HGP Goal to sequence the whole human genome They succeeded Gene mapping then physical mapping the DNA sequencing http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml