DNA Technology and Genomics Chapter 12 DNA Technology and Genomics

DNA and Crime Scene Investigations Many violent crimes go unsolved lack of enough evidence If biological fluids are left at a crime scene DNA can be isolated from them

DNA fingerprinting: set of laboratory procedures determines w/near certainty whether 2 samples of DNA are from same individual powerful tool for CSIs Investigator at one of the crime scenes (above), Narborough, England (left)

BACTERIAL PLASMIDS AND GENE CLONING 12.1 Plasmids are used to customize bacteria: An overview Gene cloning is 1 application DNA technology Methods for studying & manipulating genetic material

And insert those plasmids into bacteria Researchers can insert desired genes into plasmids, creating recombinant DNA And insert those plasmids into bacteria Figure 12.1

Bacterium Bacterial Plasmid chromosome Figure 12.1 Plasmid isolated Cell containing gene of interest 2 DNA isolated Bacterial chromosome 3 Gene inserted into plasmid Plasmid DNA Recombinant DNA (plasmid) Gene of interest 4 Plasmid put into bacterial cell Recombinant bacterium Gene for pest Resistance inserted into plants 5 Cell multiplies with gene of interest Copies of gene Copies of protein Clone of cells Protein used to make snow form at higher temperature Figure 12.1 Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy

If the recombinant bacteria multiply into a clone The foreign genes are also copied

12.2 Enzymes are used to “cut and paste” DNA The tools used to make recombinant DNA are Restriction enzymes, which cut DNA at specific sequences DNA ligase, which “pastes” DNA fragments together

Restriction enzyme recognition sequence Creating recombinant DNA using REs & DNA ligase DNA G A A T T C C T T A A G 1 Restriction enzyme recognition sequence Restriction enzyme cuts the DNA into fragments G A AT TC C T T A A 2 G Sticky end A AT TC G Addition of a DNA fragment from another source G 3 C T T A A Two (or more) fragments stick together by base-pairing G A AT T C C T TA A G G A AT T C C T TA A G 4 DNA ligase pastes the strand Recombinant DNA molecule 5 Figure 12.2

12.3 Genes can be cloned in recombinant plasmids: A closer look 12.3 Genes can be cloned in recombinant plasmids: A closer look Bacteria take the recombinant plasmids from their surroundings reproduce cloning the plasmids and the genes they carry

Cloning a gene in a bacterial plasmid 1 Isolate DNA from two sources E.coli Human cell DNA 2 Cut both DNAs with the same restriction enzyme Plasmid Gene V Sticky ends 3 Mix the DNAs; they join by base-pairing 4 Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid Gene V 5 Put plasmid into bacterium by transformation Recombinant bacterium 6 Clone the bacterium Figure 12.3 Bacterial clone carrying many copies of the human gene

CONNECTION 12.6 Recombinant cells and organisms can mass-produce gene products Applications of gene cloning include medical and other uses Table 12.6

Different organisms, including bacteria, yeast, and mammals Different organisms, including bacteria, yeast, and mammals Can be used for this purpose Figure 12.6

12.7 DNA technology is changing the pharmaceutical industry CONNECTION 12.7 DNA technology is changing the pharmaceutical industry widely used to produce medicines and to diagnose diseases

Therapeutic hormones In 1982, humulin, human insulin produced by bacteria 1st recombinant drug approved by FDA Figure 12.7A

Diagnosis and Treatment of Disease Diagnosis and Treatment of Disease DNA technology Is being used increasingly in disease diagnosis

Vaccines DNA technology Vaccines DNA technology Is also helping medical researchers develop vaccines Figure 12.7B

pGLO LAB

RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING 12.10 Gel electrophoresis sorts DNA molecules by size + – Power source Gel Mixture of DNA molecules of different sizes Longer molecules Shorter Completed gel Figure 12.10

12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

How Restriction Fragments Reflect DNA Sequence How Restriction Fragments Reflect DNA Sequence Restriction fragment length polymorphisms (RFLPs) Reflect differences in the sequences of DNA samples Crime scene Suspect w x y z Cut DNA from chromosomes C G A T Figure 12.11A

After digestion by restriction enzymes fragments are run through a gel After digestion by restriction enzymes fragments are run through a gel – + Longer fragments Shorter x w y z 1 2 Figure 12.11B

Using DNA Probes to Detect Harmful Alleles Using DNA Probes to Detect Harmful Alleles Radioactive probes Can reveal DNA bands of interest on a gel

Detecting a harmful allele using restriction fragment analysis Detecting a harmful allele using restriction fragment analysis 1 2 3 4 5 Restriction fragment preparation Gel electrophoresis Blotting Radioactive probe Detection of radioactivity (autoradiography) I II III Restriction fragments Filter paper Probe Radioactive, single- stranded DNA (probe) Film Figure 12.11C

CONNECTION 12.12 DNA technology is used in courts of law

DNA fingerprinting can help solve crimes DNA fingerprinting can help solve crimes Figure 12.12B Defendant’s blood Blood from defendant’s clothes Victim’s Figure 12.12A

CONNECTION 12.13 Gene therapy may someday help treat a variety of diseases Gene therapy Is the alteration of an afflicted individual’s genes

Gene Therapy Cloned gene (normal allele) 1 Insert normal gene into virus Retrovirus Viral nucleic acid 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome Bone marrow cell from patient 4 Inject cells into patient Bone marrow Figure 12.13

Gene therapy May one day be used to treat both genetic diseases and nongenetic disorders Unfortunately, progress is slow

12.14 The PCR method is used to amplify DNA sequences 12.14 The PCR method is used to amplify DNA sequences The polymerase chain reaction (PCR) Can be used to clone a small sample of DNA quickly, producing enough copies for analysis 1 2 4 8 Initial DNA segment Number of DNA molecules Figure 12.14

GENOMICS CONNECTION 12.15 The Human Genome Project is an ambitious application of DNA technology The Human Genome Project, begun in 1990 and now largely completed Genetic and physical mapping of chromosomes, followed by DNA sequencing Figure 12.15

The data are providing insight into Development, evolution, and many diseases

12.16 Most of the human genome does not consist of genes 12.16 Most of the human genome does not consist of genes The haploid human genome contains about 25,000 genes And a huge amount of noncoding DNA

Much of the noncoding DNA consists of repetitive nucleotide sequences And transposons that can move about within the genome

12.17 The science of genomics compares whole genomes CONNECTION 12.17 The science of genomics compares whole genomes The sequencing of many prokaryotic and eukaryotic genomes

Besides being interesting themselves... Nonhuman genomes can be compared with the human genome Table 12.17

Proteomics Is the study of the full sets of proteins produced by organisms

GENETICALLY MODIFIED ORGANISMS: CONNECTION 12.18 Genetically modified organisms are transforming agriculture

gene within plant chromosome Recombinant DNA technology Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms Agrobacterium tumefaciens DNA containing gene for desired trait Plant cell Ti plasmid 1 Insertion of gene into plasmid using restriction enzyme and DNA ligase 2 Introduction into plant cells in culture 3 Regeneration of plant Recombinant Ti plasmid T DNA carrying new gene within plant chromosome T DNA Plant with new trait Restriction site Figure 12.18A

have had genes from other organisms inserted into their genomes Transgenic organisms have had genes from other organisms inserted into their genomes Figure 12.18B

A number of important crops and plants Are genetically modified

12.19 Could GM organisms harm human health or the environment? CONNECTION 12.19 Could GM organisms harm human health or the environment? Development of GM organisms Requires significant safety measures Figure 12.19A

Genetic engineering involves risks Genetic engineering involves risks Such as ecological damage from GM crops Figure 12.19B

CONNECTION 12.20 Genomics researcher Eric Lander discusses the Human Genome Project Genomics pioneer Eric Lander Points out that much remains to be learned from the Human Genome Project Figure 12.20

12.17 The science of genomics compares whole genomes 12.17 The science of genomics compares whole genomes The sequencing of many prokaryotic and eukaryotic genomes Has produced data for genomics, the study of whole genomes

12.4 Cloned genes can be stored in genomic libraries 12.4 Cloned genes can be stored in genomic libraries Genomic libraries, sets of DNA fragments containing all of an organism’s genes Can be constructed and stored in cloned bacterial plasmids or phages Recombinant plasmid Genome cut up with restriction enzyme Recombinant phage DNA or Bacterial clone Phage clone Phage library Plasmid library Figure 12.4

12.5 Reverse transcriptase helps make genes for cloning 12.5 Reverse transcriptase helps make genes for cloning Reverse transcriptase can be used to make smaller, complementary DNA (cDNA) libraries Containing only the genes that are transcribed by a particular type of cell Cell nucleus DNA of eukaryotic gene Exon Intron Exon Intron Exon 1 Transcription RNA transcript 2 RNA splicing (removes introns) mRNA 3 Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand Test tube Reverse transcriptase cDNA strand 4 Breakdown of RNA 5 Synthesis of second DNA strand cDNA of gene (no introns) Figure 12.5

RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING 12.8 Nucleic acid probes identify clones carrying specific genes DNA technology methods Can be used to identify specific pieces of DNA

Can tag a desired gene in a library A nucleic acid probe Is a short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA Can tag a desired gene in a library Radioactive probe (DNA) Single-stranded DNA Mix with single- stranded DNA from various bacterial (or phage) clones Base pairing indicates the gene of interest A T C C G A A T G C G C T T A T C G A G C C T T A T G C A T A T C C G A A G G T A G G C T A A Figure 12.8

12.9 DNA microarrays test for the expression of many genes at once CONNECTION 12.9 DNA microarrays test for the expression of many genes at once DNA microarray assays Can reveal patterns of gene expression in different kinds of cells

DNA microarray Figure 12.9 DNA microarray Each well contains DNA DNA microarray DNA microarray Each well contains DNA from a particular gene Actual size (6,400 genes) 1 mRNA isolated 4 Unbound cDNA rinsed away Reverse transcriptase and fluorescent DNA nucleotides Fluorescent spot Nonfluorescent spot 3 cDNA applied to wells 2 cDNA made from mRNA cDNA DNA of an expressed gene DNA of an unexpressed gene Figure 12.9