Chapter 12 DNA Technology and the Human Genome

Do Now: 10 minutes Study Technique Skim chapter opening pg 230-231 1) Define bold words and look at pictures 2) Make a quick outline of main points with at least 3 spaces in between in one color 3) Use a different color to fill in under each section with details from the book 4) In a different color come up with two questions, one easy and one difficult

Should look like this…. Recombinant DNA technology: combining genes from different sources DNA technology: methods for studying and manipulating DNA Genomics: Study of genomes based on sequence Bioinformatics: using math to analyze bio data Proteomics: study of proteins Bacteria studies Joined by pilli (male) passing DNA to “female” Respond to environment Lederburg and Tatum: experiments that mixed genes in E. coli (Recombinant DNA technology DNA technology Introns discovered by this Lead in to HGP Human Genome project (more than just humans) Mapping genomes lead to genomics, bioinformatics, and proteomics Scientists engineer bacteria for practical purposes (make pesticides, cancer drugs, AIDS vaccine) Ethical issues on health

What might Ms. Bell Ask… Easy: How do bacteria have sex? Hard: Why is this advantageous for bacteria? Easy: Describe the difference between genomics, proteomics, and bioinformatics Hard: Why are people concerned with recombinant DNA technology? Explain a hypothetical situation.

12.1 In nature, bacteria can transfer DNA in three ways Gene transfer DNA from one to another bacteria Non-reproductive – new combination DNA= closed loop w/protein Three mechanisms 1) Transformation: uptake from surrounding Griffith experiment: harmless bacteria take up piece from deadly and became deadly…. 2) Transduction: phage 3) Conjugation: Male donor, pili Female recipient Cytoplasmic bridge New DNA may integrate into recipient

Question… Which type of gene transfer allows one copy of DNA to peel off and transfer to recipient?

12.2 Bacteria plasmids can serve as carriers for gene transfer F factor Genes for conjugation Also contains origin of replication Acts as the leading end of transferred chromosome Plasmid Small circular DNA that contains origin of replication Some contain F factor Vector s: Can carry genes other than F factor R plasmid Contain enzymes that destroy antibiotics like penicillin or tetracycline Act as useful vectors in genetic engineering

How restriction enzymes work… LE 12-02 Restriction enzyme recognition sequence G A A T T C How restriction enzymes work… DNA C T T A A G Restriction enzyme cuts the DNA into fragments G A A T T C C T T A A G Sticky end A A T Addition of a DNA fragment from another source T C G G C T T A A Two (or more) fragments stick together by base-pairing G A A T T C G A A T T C C T T A A G C T T A A G DNA ligase pastes the strand Recombinant DNA molecule

LE 12-01-3 Bacterium Plasmid isolated Cell containing gene of interest DNA isolated Gene inserted into plasmid Bacterial chromosome Plasmid Recombinant DNA (plasmid) Gene of interest DNA Plasmid put into bacterial cell Recombinant bacterium Cell multiplies with gene of interest Copies of gene Copies of protein Clone of cells Gene for pest resistance inserted into plants Protein used to make snow form at higher temperature Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy

12.3 Plasmids are used to customize bacteria: An overview 1) plasmid isolated 2) DNA of interest is obtained 3) DNA inserted 4) Bacteria uptakes plasmid 5) genetically engineered, recombinant bacteria cloned Challenge: How can R plasmids be helpful in terms of recombinant DNA technology?

LE 12-03 E. coli Human cell Isolate DNA from two sources Cut both DNAs with the same restriction enzyme Plasmid DNA Gene V Sticky ends Mix the DNAs; they join by base-pairing Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid Gene V Put plasmid into bacterium by transformation Recombinant bacterium Clone the bacterium Bacterial clone carrying many copies of the human gene

Genome cut up with restriction enzyme Recombinant plasmid Recombinant LE 12-04 Genome cut up with restriction enzyme Recombinant plasmid Recombinant phage DNA or Bacterial clone Phage clone Plasmid library Phage library

12.7 Reverse Transcriptase helps make genes for cloning Do Now: Turn to page 236 section 12.7 1) Read the section 2) Redraw the picture 3) Describe cDNA and the process of making a cDNA 4) Why might cDNA of insulin might work better in E.coli than trying to use a normal gene?

LE 12-05 Cell nucleus Exon Intron Exon Intron Exon DNA of eukaryotic gene Transcription RNA transcript RNA splicing (removes introns) mRNA Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand Test tube Reverse transcriptase cDNA strand Breakdown of RNA Synthesis of second DNA strand cDNA of gene (no introns)

12.8 Nucleic acid probes identify clones carrying specific genes It is difficult to tell if clone contains ________________________ Methods depends on base pairing Part of the DNA is known Gene _________ Probe (radioactive) _________ Steps: 1) _________________________________ 2) Treat (heat or alkali) to ___________________ 3) Add radioactive probe (reality ___________________) 4)Autoradiography (develop film to determine radioactivity) 5) Compare to master plate (____________________)

Radioactive probe (DNA) A A T C C G Mix with single- stranded DNA from various bacterial (or phage) clones Single-stranded DNA G C T A A G T T C T A C C C A G T A G C T A G T C T A G A T G A T T C G Base pairing indicates the gene of interest C G G A A

12.9 DNA microarrays test for expression of many genes DNA microarrays (chips) Used for______________________________ Measures gene expression at _______________ Steps ______________________ mRNA used to make radioactive ________ Exposed to a chip Benefits Thousands of genes tested at once Each radioactive spot represents gene expression

LE 12-09 DNA microarray Each well contains DNA from a particular gene Actual size (6,400 genes) mRNA isolated Unbound cDNA rinsed away Reverse transcriptase and fluorescent DNA nucelotides Fluorescent spot Nonfluorescent spot cDNA applied to wells cDNA made from mRNA cDNA DNA of an expressed gene DNA of an unexpressed gene

12.10- 12.11 Gel electrophoresis: Mixture of DNA molecules of different sizes Longer molecules Power source Gel Shorter molecules Completed gel

12.12 The PCR method is used to amplify DNA sequences Polymerase chain reaction Steps: _____________________ DNA is incubated with____________________________________________ Benefits: Each cycle doubles DNA amount Takes hours Can copy specific segment Starting material does not need to be purified

PCR: The good and the bad Even though it is faster, it cannot replace gene cloning in cells due to occasional errors that impose limits Uses: ________________________

12.13 Most of the human genome does not consist of genes Most of our DNA does not code for protein 35,000 genes that code for protein, tRNA and rRNA 97% is non-coding

Types of junk __________and DNA between genes Repetitive DNA Short repeats Associated with ________________and ends of chromosomes (chromosome structure) Telomeres: at the end of chromosomes and related to cell death and cancer Cells that can regenerate telomeres can “_______________” Might deal with gene ___________________ Some nervous diseases caused by abnormal stretches of triplet Huntington's: _____ repeat in coding region Long Repeats: function unknown Example: Transposons: genes that can jump from one location to the next (researcher Barbara McClintock) Cut and paste or ______, cut and paste (leaves sequence behind) Natural mutagen to generate ________________ Implicated in some cancer

LE 12-11b 1 2 Longer fragments z x w Shorter fragments y y

Blood from defendant’s clothes Defendant’s blood Victim’s blood LE 12-12a Blood from defendant’s clothes Defendant’s blood Victim’s blood

LE 12-13 Cloned gene (normal allele) Insert normal gene into virus Viral nucleic acid Retrovirus Infect bone marrow cell with virus Viral DNA inserts into chromosome Bone marrow cell from patient Bone marrow Inject cells into patient

LE 12-14 Initial DNA segment 1 2 4 8 Number of DNA molecules

12.4-12.6 Recombinant Lab Add picture from each….