DNA Technology & Genomics Chapter 20

Biotechnology Today Genetic Engineering Our tool kit… manipulation of DNA if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with this unit is a survey of those tools… Our tool kit…

Understanding and Manipulating Genomes DNA sequencing has depended on advances in technology, starting with making recombinant DNA Recombinant DNA - nucleotide sequences from 2 different sources are combined in vitro into same DNA molecule Methods for making recombinant DNA are central to genetic engineering, direct manipulation of genes for practical purposes

20.1: DNA cloning permits production of multiple copies of a specific gene To work with specific genes, scientists prepare gene-sized pieces of DNA in identical copies - gene cloning

Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes - cut DNA molecules at DNA sequences called restriction sites Restriction enzyme usually makes many cuts, yielding restriction fragments Most useful restriction enzymes cut DNA in a staggered way - producing fragments with “sticky ends” - bond with complementary “sticky ends” of other fragments DNA ligase - enzyme that seals bonds between restriction fragments

One possible combination Recombinant DNA molecule Restriction site DNA 5¢ 3¢ 3¢ 5¢ Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Sticky end DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme One possible combination DNA ligase seals the strands. Recombinant DNA molecule

Cloning a Eukaryotic Gene in a Bacterial Plasmid In gene cloning, original plasmid is called a cloning vector Cloning vector - DNA molecule that can carry foreign DNA into a cell and replicate there

Producing Clones of Cells Cloning human gene in bacterial plasmid: 1. Vector and gene-source DNA are isolated 2. DNA is inserted into vector 3. Human DNA fragments are mixed with cut plasmids, and base-pairing takes place 4. Recombinant plasmids are mixed with bacteria 5. The bacteria are plated and incubated 6. Cell clones with the right gene are identified

Cut both DNA samples with the same restriction enzyme. Human DNA Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Cut both DNA samples with the same restriction enzyme. Human DNA fragments Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids

LE 20-4_2 Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Cut both DNA samples with the same restriction enzyme. Human DNA fragments Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria

LE 20-4_3 Human DNA fragments Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Cut both DNA samples with the same restriction enzyme. Human DNA fragments Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone

Identifying Clones Carrying a Gene of Interest Clone carrying gene of interest can be identified with a nucleic acid probe Called nucleic acid hybridization Radioactive or fluorescent probes are engineered to be complimentary to a target sequence First, denature of cells’ DNA

LE 20-5 Colonies containing gene of interest Master plate Probe DNA Radioactive single-stranded DNA Solution containing probe Gene of interest Filter Single-stranded DNA from cell Film Filter lifted and flipped over Hybridization on filter A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

Storing Cloned Genes in DNA Libraries Genomic library - collection of recombinant vector clones produced by cloning DNA fragments from an entire genome

Complementary DNA (cDNA) library - made by cloning DNA made in vitro by reverse transcription of all mRNA produced by a particular cell cDNA library - represents only part of genome—only subset of genes transcribed into mRNA in original cells Solves problem of prokaryotes not having machinery to remove introns

Bacterial Expression Systems Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells Have to overcome differences in promoters and other DNA control sequences

Eukaryotic Cloning and Expression Systems Use of yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems YACs - behave normally in mitosis and can carry more DNA than a plasmid Eukaryotic hosts can provide posttranslational modifications that many proteins require

Introducing recombinant DNA into eukaryotic cells: electroporation, - applying a brief electrical pulse to create temporary holes in plasma membranes inject DNA into cells using microscopic needles

Polymerase Chain Reaction (PCR) Polymerase chain reaction (PCR) - produce many copies of specific target segment of DNA 3-step cycle: heating, cooling, and replication chain reaction that produces an exponentially growing population of identical DNA molecules http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

LE 20-7 5¢ 3¢ Target sequence Genomic DNA 3¢ 5¢ Denaturation: Heat briefly to separate DNA strands 5¢ 3¢ 3¢ 5¢ Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Cycle 1 yields 2 molecules Primers Extension: DNA polymerase adds nucleotides to the 3¢ end of each primer New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction sites Restriction fragment analysis - detects differences in nucleotide sequences of DNA molecules provide comparative information about DNA sequences

Gel Electrophoresis and Southern Blotting One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis Uses a gel as a molecular sieve to separate nuclei acids or proteins by size DNA is negatively charged and moves towards a positive charge when placed in an electrical field

RFLP Analysis restriction fragment analysis - fragments of DNA molecule are sorted by gel electrophoresis Useful for comparing two different DNA molecules, such as two alleles for a gene

Restriction Fragment Length Differences as Genetic Markers Restriction fragment length polymorphisms (RFLPs, or Rif-lips) - differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths A RFLP can serve as genetic marker for a particular location (locus) in the genome RFLPs are detected by Southern blotting

LE 20-9 Normal b-globin allele 175 bp 201 bp Large fragment Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Large fragment Ddel Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles

Uses: Evolutionary relationships Comparing DNA samples from different organisms to measure evolutionary relationships turtle snake rat squirrel fruitfly – 1 3 2 4 5 1 2 3 4 5 DNA  +

Uses: Medical diagnostic Comparing normal allele to disease allele chromosome with normal allele 1 chromosome with disease-causing allele 2 allele 1 allele 2 – DNA  Example: test for Huntington’s disease +

Uses: Forensics Comparing DNA sample from crime scene with suspects & victim suspects crime scene sample S1 S2 S3 V – DNA  +

DNA fingerprints Comparing blood samples on defendant’s clothing to determine if it belongs to victim DNA fingerprinting comparing DNA banding pattern between different individuals ~unique patterns

Southern blotting - combines gel electrophoresis with nucleic acid hybridization Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to DNA immobilized on a “blot” of gel http://highered.mcgraw-hill.com/olc/dl/120078/bio_g.swf

DNA + restriction enzyme LE 20-10 Heavy weight DNA + restriction enzyme Restriction fragments I I I Nitrocellulose paper (blot) Gel Sponge Paper towels I Normal -globin allele I Sickle-cell allele I Heterozygote Alkaline solution Preparation of restriction fragments. Gel electrophoresis. Blotting. Probe hydrogen- bonds to fragments containing normal or mutant -globin I I I Radioactively labeled probe for -globin gene is added to solution in a plastic bag I I I Fragment from sickle-cell -globin allele Film over paper blot Fragment from normal -globin allele Paper blot Hybridization with radioactive probe. Autoradiography.

Concept 20.3: Entire genomes can be mapped at the DNA level Most ambitious mapping project to date has been the sequencing of the human genome Officially begun as Human Genome Project in 1990, sequencing was largely completed by 2003 Scientists have also sequenced genomes of other organisms, providing insights of general biological significance

Genetic (Linkage) Mapping: Relative Ordering of Markers 1st stage is constructing linkage map of several thousand genetic markers throughout each chromosome Order of markers and relative distances between them are based on recombination frequencies

LE 20-11 Cytogenetic map Chromosome bands Genes located by FISH Genetic (linkage) mapping Genetic markers Physical mapping Overlapping fragments DNA sequencing

Physical Mapping: Ordering DNA Fragments Physical map - constructed by cutting DNA molecule into many short fragments and arranging them in order by identifying overlaps Physical mapping gives actual distance in base pairs between markers

DNA Sequencing Relatively short DNA fragments can be sequenced by dideoxy chain-termination method Inclusion of special dideoxyribonucleotides in reaction mix ensures that fragments of various lengths will be synthesized http://www.dnalc.org/resources/animations/cycseq.html

LE 20-12 DNA (template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) 3¢ 5¢ 5¢ DNA polymerase 3¢ DNA (template strand) Labeled strands 5¢ 3¢ 3¢ Direction of movement of strands Laser Detector

Linkage mapping, physical mapping, and DNA sequencing represent overarching strategy of Human Genome Project An alternative approach to sequencing genomes starts with sequencing random DNA fragments Computer programs then assemble overlapping short sequences into one continuous sequence

LE 20-13 Cut the DNA from many copies of an entire chromosome into overlapping frag-ments short enough for sequencing Clone the fragments in plasmid or phage vectors Sequence each fragment Order the sequences into one overall sequence with computer software

Concept 20.4: Genome sequences provide clues to important biological questions In genomics, scientists study whole sets of genes and their interactions Genomics is yielding new insights into genome organization, regulation of gene expression, growth and development, and evolution

Identifying Protein-Coding Genes in DNA Sequences Computer analysis of genome sequences helps identify sequences likely to encode proteins The human genome contains about 25,000 genes, but the number of human proteins is much larger Comparison of sequences of “new” genes with those of known genes in other species may help identify new genes NOVA Science Now: Public Genomes

Determining Gene Function One way to determine function is to disable gene and observe consequences Using in vitro mutagenesis, mutations are introduced into cloned gene, altering or destroying its function When mutated gene is returned to cell, normal gene’s function might be determined by examining the mutant’s phenotype In nonmammalian organisms, a simpler and faster method, RNA interference (RNAi), has been used to silence expression of selected genes

Studying Expression of Interacting Groups of Genes Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays DNA microarray assays - compare patterns of gene expression in different tissues, at different times, or under different conditions

LE 20-14 Tissue sample Isolate mRNA. mRNA molecules Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Labeled cDNA molecules (single strands) DNA microarray Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. Size of an actual DNA microarray with all the genes of yeast (6,400 spots)

Comparing Genomes of Different Species Comparative studies of genomes from related and widely divergent species provide information in many fields of biology The more similar the nucleotide sequences between two species, the more closely related these species are in their evolutionary history Comparative genome studies confirm the relevance of research on simpler organisms to understanding human biology NOVA Science NOW: Autism Video

Future Directions in Genomics Genomics - study of entire genomes Proteomics - systematic study of all proteins encoded by a genome Single nucleotide polymorphisms (SNPs) - provide markers for studying human genetic variation

Concept 20.5: The practical applications of DNA technology affect our lives in many ways Many fields benefit from DNA technology and genetic engineering

Medical Applications One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases Gene testing videohttp://www.pbs.org/wgbh/nova/body/public-genomes.html

Diagnosis of Diseases Scientists can diagnose many human genetic disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation Even when a disease gene has not been cloned, presence of an abnormal allele can be diagnosed if a closely linked RFLP marker has been found

RFLP marker Disease-causing allele Normal allele DNA Disease-causing allele Restriction sites Normal allele

Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of genes into cells Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

LE 20-16 Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Bone marrow Inject engineered cells into patient.

Pharmaceutical Products Some pharmaceutical applications of DNA technology: Large-scale production of human hormones and other proteins with therapeutic uses Production of safer vaccines

Forensic Evidence DNA “fingerprints” obtained by analysis of tissue or body fluids can provide evidence in criminal and paternity cases A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel The probability that two people who are not identical twins have the same DNA fingerprint is very small Exact probability depends on the number of markers and their frequency in the population

Blood from defendant’s LE 20-17 Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V)

Environmental Cleanup Genetic engineering can be used to modify the metabolism of microorganisms Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials

Agricultural Applications DNA technology is being used to improve agricultural productivity and food quality

Animal Husbandry and “Pharm” Animals Transgenic organisms are made by introducing genes from one species into the genome of another organism Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles) Other transgenic organisms are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use

Genetic Engineering in Plants Agricultural scientists have endowed a number of crop plants with genes for desirable traits The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

Agrobacterium tumefaciens LE 20-19 Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts T DNA DNA with the gene of interest Recombinant Ti plasmid Plant with new trait

Safety and Ethical Questions Raised by DNA Technology Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures Most public concern about possible hazards centers on genetically modified (GM) organisms used as food