Ap Bio Outline

Chapter 20 Outline

20.1 DNA cloning yields multiple copies of a gene or other DNA segment

1. Key terms

1. Recombinant DNA: DNA molecules formed when segments of DNA from two different courses are combined in vitro (in test tube)
2. Biotechnology: manipulation of organisms or their components to make useful products
3. Genetic engineering: direct manipulation of genes for practical purposes

1. DNA Cloning and Its Applications

1. Plasmids: small circular DNA molecules that replicate separately from the bacterial chromosome

1. Researchers obtain a plasmid originally isolated from bacterial cell and insert “foreign” DNA into it -> recombinant DNA molecule
2. Plasmid with recombinant DNA molecule is then returned to bacterial cell -> recombinant bacterium

1. Gene cloning: using bacteria to make multiple copies of a gene

1. Amplifies a particular gene and to produce a protein product

1. Using Restriction Enzymes to Make Recombinant DNA

1. Restriction enzymes cut DNA at specific restriction sites (DNA sequences)
2. A restriction enzyme usually makes many cuts, yielding restriction fragments

1. Cut DNA in staggered way, producing “sticky ends” (single stranded end; can form hydrogen-bonded base pairs with complementary sticky ends on any other DNA molecules cut with same restriction enzyme)
2. Can be used on genes of interest as well
3. DNA ligase seals the bonds between restriction fragments
4. All copies of a particular DNA molecule always yield the same set of restriction fragments when exposed to the same restriction enzyme

1. Cloning a Eukaryotic Gene in a Bacterial Plasmid

1. Cloning vector: DNA molecule that can carry foreign DNA into host cell and replicate there

1. Plasmids are widely used as cloning vectors

1. Can be readily obtained from commercial suppliers
2. Can be manipulated to from recombinant plasmids by insertion of foreign DNA in vitro and then introduced into bacterial cells
3. Recombinant bacterial plasmids (and foreign DNA they carry) can multiply rapidly

1. Producing Clones of Cells Carrying Recombinant Plasmids

1. Hummingbird genes inserted into plasmids from E. Coli

1. Isolate hummingbird genomic DNA and obtain bacterial plasmid from E. Coli, which carries ampR (resistant to ampicillin) and lacZ (encodes β-galactosidase and hydrolyzes X-gal to form a blue product)
2. Plasmid only contains one copy of restriction site recognized by a particular restriction enzyme, which is then used to cut both the plasmid and hummingbird DNA
3. Fragments are mixed together and join by base pairing (DNA ligase seals); resulting recombinant plasmids may contain single hummingbird DNA fragments and some carry all or part of β-globin gene
4. DNA mixture is then added to bacteria that have a mutation in the lacZ gene; some cells acquire a recombinant plasmid carrying a gene, some take nonrecombinant plasmid (fragment of noncoding hummingbird DNA), or nothing at all
5. Plating out all bacteria on solid nutrient medium containing ampicillin allows distinguishing of whether the cells took up plasmids or not (only cells with a plasmid will reproduce because they have ampR gene, allowing resistance to the antibiotic ampicillin)
6. Presence of X-gal in medium allows distinguishing between colonies with recombinant plasmids and those with nonrecombinant plasmids (colonies with nonrecombinant plasmids have lacZ gene and can produce functional β-galactosidase, effectively hydrolyzing X-gal and forming a blue product)
7. RESULTS OF EXPERIMENT: only a cell that took up a plasmid (contains ampR gene) will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue (can hydrolyze X-gal). Colonies with recombinant plasmids (with disrupted lacZ gene) will be white (cannot hydrolyze X-gal)

*isolate target DNA and a bacterial plasmid, and use a restriction enzyme to cut both up at the same restriction sites. Mix the fragments together and use DNA ligase to bind together. Return these recombinant DNA to the bacterium (later you can “infect” the target organism with the recombinant bacterium)

1. Storing Cloned Genes in DNA Libraries

1. “shotgun approach”: no single gene is targeted for cloning
2. Genomic library: complete set of recombinant plasmid clones, each carrying copies of a particular segment from the initial genome
3. Bacterial artificial chromosome (BAC): large plasmids, trimmed down so they contain just the genes necessary to ensure replication
4. Clones are usually stored in multiwelled plastic plates (one clone per well)
5. Making complementary DNA (cDNA) from eukaryotic genes

1. Reverse transcriptase added to test tube containing isolated mRNA
2. Reverse transcriptase makes first DNA strand using mRNA template and stretch of dT’s (thymine deoxyribonucleotides) as a DNA primer
3. mRNA is degraded by another enzyme
4. DNA polymerase synthesizes second strand, using primer in reaction mixture
5. Result is cDNA, which carries complete coding sequence of the gene but no introns

1. cDNA can be inserted into vector DNA; extracted mRNA is mixture of all mRNA molecules in original cells, transcribed from many different genes
2. cDNA library: made up of cDNAs that are cloned; represents only part of the genome (only the subset of genes that were transcribed in the cells from which mRNA was isolated)
3. Genomic library is good for if you want to clone a gene but don’t know what cell type expresses it or cannot obtain enough cells of the appropriate type, or studying about regulatory sequences and introns associated with a gene
4. cDNA library is good for studying a specific protein or used to study sets of genes expressed in particular cell types

1. Screening a Library for Clones Carrying a Gene of Interest

1. Nucleic acid hybridization: determines gene’s DNA’s ability to base-pair with a complementary sequence on another nucleic acid molecule
2. Nucleic acid probe: complementary molecule (short single-stranded RNA or DNA)
3. Each probe molecule (labeled with radioactive isotope) will hydrogen-bond specifically to a complementary sequence in desired gene
4. For a radioactive probe, the location of the black spot on a piece of photographic film identifies the clone containing the gene of interest

1. Bacterial Expression Systems

1. Expression vector: cloning vector that contains highly active bacterial promoter just upstream of a restriction site where the eukaryotic gene can be inserted in correct reading frame
2. Bacterial host will recognize the promoter (from the expression vector) and proceed to express the foreign gene
3. Another issue is that bacteria do not have RNA-splicing machinery, so cDNA form of a eukaryotic gene is used (only includes exons)

1. Eukaryotic Cloning and Expression Systems

1. Many eukaryotic proteins will not function unless they are modified after translation
2. Electroporation: brief electrical pulse applied to a solution containing cells creates temporary holes in their plasma membranes, through which DNA can enter

1. Cross-Species Gene Expression and Evolutionary Ancestry

1. Pax-6 gene controls gene expression of single lens and compound lens in eyes

1. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)

1. PCR can make billions of copies of a target segment of DNA in a few hours

1. PCR requires double stranded DNA containing target sequence, a heat-resistant DNA polymerase, all four nucleotides, and two 15- to 20-nucleotide DNA strands that serve as primers (each complementary to each end)

1. Denaturation: heat briefly to separate DNA strands
2. Annealing: cool to allow primers to form hydrogen bonds with ends of target sequence
3. Extension: DNA polymerase adds nucleotides to 3’ end of each primer

1. With each successive cycle, number of target segment molecules of the correct length doubles

1. PCR amplification cannot substitute for gene cloning in cells when large amounts of a gene are desired (PCR errors impose limits)

20.2 DNA technology allows us to study the sequence, expression, and function of gene

1. Gel Electrophoresis and Southern Blotting

1. Gel (made from a polymer) acts as a molecular sieve to separate nucleic acids or proteins on basis of size, electrical charge, and other physical properties
2. Nucleic acid molecules carry negative charges on their phosphate groups → travel toward the positive pole in an electric field

1. As they move, agarose fibers impede impede longer molecules more than it does shorter ones (therefore shorter ones can travel faster), separating them by length (separates into bands)
2. After current is turned off, a DNA-binding dye is added to reveal the separated bands

1. Restriction fragment analysis: DNA fragments produced by restriction enzyme digestion are separated by gel electrophoresis, yielding a band pattern (can be used to compare two different DNA molecules ex. two alleles of a gene)
2. Polymorphisms: variations in DNA sequence among a population

1. Restriction fragment length polymorphism (RFLP): change in one base pair of a sequence in restriction site
2. If one allele contains a RFLP, digestion with the enzyme that recognizes the site will produce a different mixture of fragments for each of the two alleles, hence yielding a different band pattern

1. Southern blotting: combines gel electrophoresis and nucleic acid hybridization

1. Probe used is usually a labeled single-stranded DNA molecule that is complementary to the gene of interest

1. Each DNA sample is mixed with same restriction enzyme, such as DdeI. Digestion of each sample yields a mixture of restriction fragments
2. Restriction fragments in each sample are separated by electrophoresis
3. Capillary action pulls alkaline solution upward through the gel, denaturaing and transferring the DNA to a nitrocellulose membrane (producing a blot)
4. Nitrocellulose blot is exposed to a solution containing a probe
5. A sheet of photographic film is laid over the blot, in which the radioactivity in the bound probe exposes the film to form an image corresponding to those bands containing DNA that base-paired with the probe

1. DNA Sequencing

1. Dideoxyribonucleotide chain termination method synthesize a set of DNA strands complementary to original DNA fragment

1. Each strand starts with same primer and ends with a dideoxyribonucleotide (ddNTP), which terminates a growing DNA strand (lacks 3’ –OH group)
2. Each type of ddNTP is tagged with a distinct flourescent label

1. Fragment of DNA is denatured into single strands; primer, DNA polymerase, 4 ddNTPs (each tagged with specific fluorescent molecule), and 4 DNA nucleotides added
2. Synthesis of each new strand starts at 3’ end of primer and continues until ddNTP is inserted at random, which terminates further elongation of strand (set of labeled strands of various lengths is generated, with color of tag representing last nucleotide in sequence)
3. Labeled strands in mixture are separated by passage through a polyacrylamide gel, with shorter strands moving through more quickly. Fluorescent detector senses color of each fluorescent tag as strands come through (color of each tag on each strand indicates identity of the nucleotide at its end)

1. Analyzing Gene Expression

1. Studying the Expression of Single Genes

1. Northern blotting: carry out gel electrophoresis on samples of mRNA on embryos at different stages of development, transfer samples to a nitrocellulose membrane, then allow the mRNAs on the membrane to hybridize with a labeled probe

1. Can be used to hypothesize when a protein functions in stages of development

1. Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive (requires less mRNA)

1. Isolation of mRNAs from different developmental starges of embryos
2. cDNA synthesis is carried out by incubating the mRNAs with reverse transcriptase
3. PCR amplification of sample is performed using primers specific to the gene
4. Gel electrophoresis will reveal amplified DNA products only in samples that contained mRNA transcribed from specific gene

1. In situ hybridization: track down location of specific mRNAs using labeled probes in the intact organism

1. Studying the Expression of Interacting Groups of Genes

1. DNA microarrays: consists of tiny amounts of large number of single-stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array (microarray is also called a DNA chip)

1. Apply cDNA mixture (tagged with fluorescent labels) to a microarray with a different gene in each spot (cDNA hybridizes with any complementary DNA)

1. Determining Gene Function

1. In vitro mutagenesis: specific mutations are introduced into a cloned gene, and the mutated gene is returned to a cell (disables normal cellular copies of same gene)

1. Phenotype of mutant cell may help reveal function of missing normal protein
2. Ethical issues in humans prohibit knocking out genes to determine their functions

1. RNA interference (RNAi): uses synthetic double-stranded RNA molecules matching the sequence of a particular gene to trigger breakdown of the gene’s messenger RNA or to block its translation
2. Genome-wide association studies: analyze genomes of large numbers of people with certain phenotypic condition or disease to try to find differences they all share

1. Test for genetic markers (DNA sequences that vary in the population)

1. Single nucleotide polymorphism (SNP): single base-pair site where variation is found in at least 1% of the population

1. SNP itself doesn’t contribute to disease, most are located in noncoding regions
2. Marker and gene will almost always be inherited together (close proximity)

20.3 cloning organisms may lead to production of stem cells for research and other applications

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