Bacterial Genetics & DNA Technology PDF

BACTERIAL GENETICS & DNA TECHNOLOGY stranded DNA molecule (several million base pairs in length) I. BACTERIAL GENETICS o Some have multiple chromosomes...

BACTERIAL GENETICS & DNA TECHNOLOGY stranded DNA molecule (several million base pairs in length) I. BACTERIAL GENETICS o Some have multiple chromosomes o Some have linear chromosomes BACTERIAL GENOME Most bacterial chromosomes consist mainly of sequences that encode proteins haploid o 90% of E.coli DNA encodes proteins most consist of a single chromosome (some o Only 1% of human DNA encodes proteins have multiple) Plasmid Chromosome Plasmids Many bacteria possess plasmids - small, usually ▪ circular (some ▪ small, circular DNA linear) molecules (several circular DNA molecules (several thousand base thousand bps long) pairs in length) ▪ contains a ▪ carry genes not o Some are present in many copies per cell single double- essential to bacterial o Some are present in only 1 or 2 copies stranded DNA function Carry genes not essential to bacterial function molecule o roles in life o But have roles in life cycle and growth of (several cycle and bacteria million bps growth o Promote mating between bacteria long) o antiobiotic o Contain genes that kill other bacteria resistance Responsible for the spread of antibiotic ▪ consist mainly ▪ has an origin of resistance among bacteria of sequences replication that encode o independent Each possess an origin of replication proteins replication from o A specific DNA sequence where DNA chromosome replication is initiated Allows plasmid to replicate independently from bacterial chromosome GENE TRANSFER IN BACTERIA Asexual Reproduction in Bacteria o binary fission = a cell splits into two o no DNA exchange o daughter cells are genetically identical to e/o and to the parent cell Bacterial Genome Sexual Reproduction in Eukaryotes The bacterial genome is haploid and o meiosis = a diploid parent cell divides into observation of phenotypes is difficult due to haploid progeny small size of bacteria = require special methods o homologous chromosomes exchange DNA for analysis o genetically diverse offspring o Haploid = a single copy of each gene is present Chromosome Most bacterial cells possess one circular chromosome containing a single double-  2 CATEGORIES OF GENE TRANSFER 1. Vertical Gene Transfer ▪ genetic info passed from one generation to the next (parent cell to offspring) o between members of the same species ▪ whole genome is transferred ▪ via sexual or asexual reproduction Vertical Transfer of genes from parent to offspring (daughter cells) Genetic information is transferred from one generation to the next So how do bacteria achieve genetic variation? Between members of the same species E.g. sexual and asexual reproduction - organisms pass some or all their genome onto their progeny Genetic exchange in bacteria VS diploid eukaryotic sexual reproduction 2. Horizontal Gene Transfer DNA exchange and reproduction are not ▪ genetic info passed between cells of the same coupled in bacteria generation o Bacteria often undergo reproduction o conspecific = same species (binary fission) without receiving any o allospecific = different species DNA from another cell ▪ only fewer genes are transferred o Each daughter cell receives an exact ▪ via conjugation, transformation, or copy of the parent cell's DNA, resulting transduction (non-reproductive) in genetically identical cells. In most eukaryotes, genes are passed only SIGNIFICANCE among members of the same species through genetic diversity & speciation reproduction, that is, genes are passed from antibiotic resistance one generation to the next. xenobiotic tolerance o During meiosis, homologous ability to use new metabolites chromosomes (one from each parent) exchange genetic material through a process called crossing over or recombination. o Involves DNA exchange and recombination, leading to genetically diverse offspring. Lack of genetic variation increases the risk of extinction. Without variety, there may be no organisms that can survive a major change in the environment. o Prokaryotes have a different way to increase genetic variation. Horizontal ▪ Genes passed between cells of the same generation  ▪ genes can be passed between individual Conjugation members of different species by Genetic material is passed directly from one nonreproductive mechanisms, such as bacterium to another conjugation, transformation, and transduction. Direct cell-to-cell contact or via a bridge-like ▪ Genetic information is passed to a member of connection between two cells the same generation The donor cell passes a plasmid or transposon o Conspecific = genome transfer to the or part of the bacterial chromosome to the same species recipient cell o Allospecific = genome transfer to a Transfer is only from donor to recipient different species No reciprocal exchange of genetic material ▪ Serves a vital role in introducing genetic diversity to prokaryotes DISCOVERY ▪ Played a significant role in the evolution of Discovered by Joshua Lederberg and Edward bacteria Tatum in 1946 o A major factor in the process of Using two auxotrophic strains of E. coli speciation in bacteria o Auxotrophic = require a specific growth o One species may transfer antibiotic substance beyond the minimum resistance genes to another species required for normal metabolism o Genes conferring enhanced o Prototrophs = same nutritional pathogenicity may be transferred requirements as the parental strain, o Antibiotic resistance capable of synthesizing all necessary o Xenobiotic tolerance nutrients from minimal media for o Ability to use new metabolites growth. o Strain A = required methionine (met) 3 MECHANISMS OF HORIZONTAL GENE TRANSFER IN and biotin (bio) to grow BACTERIA o Strain B = required threonine (thr), leucine (leu), and thiamine (thi) A. CONJUGATION o Alone, neither of the two grew on ▪ genetic material directly passed from one minimal medium: each strain required bacterium (donor) to another (recipient) nutrients that were absent. o direct cell-to-cell contact o no reciprocal exchange between donor and recipient Discovery by Lederberg & Tatum (1946) ▪ used 2 auxotrophic strains of E. coli o auxotrophic = need additional growth substances (cannot grow in minimal media) o phototrophic = can synthesize all necessary nutrients from minimal media Strain A & B grown separately (control) ➜ no prototrophs Strain A & B mixed and grown together ➜ presence of prototrophs Conclusion: Production of prototrophs is a result of genetic transfer and recombination between the 2 auxotrophic strains.  Each strain was first grown separately Cells from both were mixed and grown together for several generations, and then plated on minimal medium o Any cells that grew were prototrophs They found that colonies of prototrophic bacteria grew on the minimal medium (genotype thr+ leu+ thi+ bio+ phe+ cys+) No prototrophs were recovered in the controls (separate plating of cells from strains A and B on minimal medium) o If mutations were responsible for the prototrophic colonies, then some colonies should also have grown on the plates containing strain A or B alone, MECHANISM but no bacteria grew on those plates. F+ cells (F for “fertility) = cells that serve as o Multiple simultaneous mutations (thr− donors of part of their chromosomes → thr+, leu− → leu+, and thi− → thi+ in F- cells = recipient bacteria, receive the donor strain Y10 or bio− → bio+, phe− → chromosome material and recombine it with phe+, and cys− → cys+ in strain Y24) part of their own chromosome would have been required for either o Conjugation can take place only strain to become prototrophic by between a cell that possesses the F mutation, which was very improbable factor and a cell that lacks the F factor. Concluded that some type of genetic transfer and recombination had taken place Bernard Davis & the Davis U-tube Base of tube has a glass filter with a pore size that allows passage of the liquid medium but is F for “fertility” too small to allow passage of bacteria. The F+ cells are placed on one side of the filter ▪ F+ cells = donates part of their chromosomes and F- cells on the other side. ▪ F- cells = receives the donated material The medium is shared by both sets of bacterial cells during incubation Bernard Davis’ U-tube Samples from both sides of tube were plated on central glass filter allows passage of medium, minimal medium = no prototrophs but not bacterial cells o Conclusion: physical contact between samples from both sides plated on minimal cells of the 2 strains is essential to medium ➜ no prototrophs genetic recombination Conclusion: Physical contact between cells of the 2 F pilus / Sex pilus strains is essential to genetic recombination. 6-9 nm tubular extension of cell membrane makes contact with receptor on F− cell ➜ pulls the cells together F factor (Fertility factor) an episome within F+ cells o episome = plasmid that can integrate into the chromosome transferred to F- cell after conjugation  consist of a circular double-stranded DNA o Many are tra genes = involved in molecule transfer of genetic info and the o contains tra genes = for transfer of formation of the F pilus genetic info and F pilus formation a copy of the F factor is almost always transferred from the F+ cell to the F- recipient, converting the recipient to the F+ state Conjugation can take place only between a cell that possesses the F factor and a cell that lacks the F factor. STEPS or PROCESS separation of the two strands of its double helix o one of the DNA strands on the F factor F pilus or sex pilus is nicked at an origin of transfer (oriT). 6- to 9-nm tubular extension of the cell One end of the nicked DNA separates membrane from the circular F plasmid and passes Mediates the process of conjugation into the recipient cell makes contact with a receptor on an F− cell, movement of one of the two strands into the contracts and pulls the two cells together. recipient cell other strand remains in the donor cell F factor (fertility factor) Both strands, one moving across the Contained within F+ cells conjugation tube and one remaining in the Consist of a circular double-stranded DNA donor cell, are replicated molecule o Replication takes place on the F factor, An autonomous genetic unit referred to as a proceeding around the circular plasmid plasmid in the F+ cell and replacing the plasmids that can exist autonomously or can transferred strand. integrate into the chromosome are further o Inside the recipient cell, the single designated as episomes strand replicates, producing a circular, o since most other plasmids are confined double-stranded copy of the F plasmid to the cytoplasm of the bacterial cell both the donor and the recipient cells become o episomes also contain an origin of F+ replication When present, the cell is able to form a sex Hfr cells (high-frequency recombination) pilus and potentially serve as a donor of special class of F+ cells genetic information F factor is integrated into the bacterial Contains as many as 40 genes chromosome donate DNA to F- cells at a high frequency F+ and F- conjugation ➜ F- becomes F+ Hfr and F- conjugation ➜ no changes to F- To become F+ or Hfr, recipient cell must receive the entire F factor or entire donor chromosome RARE EVENT since most conjugating cells break apart before the entire Hfr chromosome is transferred ❖ F− cell almost never becomes F+ or Hfr because the F factor is nicked in the middle at the initiation of strand transfer. ❖ F- cell only receives a portion of the Hfr chromosome. Conjugation between Hfr and F-cells integrated F factor is nicked, and the end of the Hfr cells (high-frequency recombination) nicked strand moves into the F− cell a special class of F+ cells But because, in an Hfr cell, the F factor has been Demonstrate an elevated frequency of integrated into the bacterial chromosome, the recombination chromosome follows the F factor into the F factor is integrated into the bacterial recipient cell. chromosome F− cell almost never becomes F+ or Hfr because If a donor cell is from an Hfr strain, recipient the F factor is nicked in the middle at the cells, though sometimes displaying genetic initiation of strand transfer, which places part recombination, never become Hfr; thus they of the F factor at the beginning and part at the remain F-. end of the strand that is transferred. To become F+ or Hfr, the recipient cell must receive the F’ cell (F prime) entire F factor, which requires that the entire donor cell containing an F plasmid with some bacterial chromosome be transferred genes happens rarely because most conjugating cells can conjugate with F- cells break apart before the entire chromosome has produces partial diploids (merozygotes) been transferred. o cells with 2 copies of some genes Since the transfer is incomplete, the F- cell only o one on the bacterial chromosome of F- receives a portion of the Hfr chromosome, cell and one on the newly introduced F which is not enough to convert it into an Hfr plasmid cell.  B. TRANSFORMATION bacterium takes up extracellular DNA from the environment via cell membrane uptake is dependent on the recipient bacterium (not all are capable or “competent”) Competence temporary state of being able to take up exogenous DNA influenced by: o growth stage o available DNA concentration o environmental conditions F prime (F’) cells Cells containing an F plasmid with some bacterial genes When an F factor is excised from the bacterial chromosome, a small amount of the bacterial chromosome may be removed with it, and these chromosomal genes will then be carried with the F plasmid F′ cells can conjugate with F− cells because F′ cells possess the F plasmid, with all the genetic information necessary for conjugation and 1. Transformation DNA transfer. Bacterium takes up extracellular or exogenous DNA from the environment or medium in which Conjugation between F’ cell and F- cell it is growing via the cell membranes the F plasmid is transferred to the F− cell, which uptake is completely dependent on the means that any genes on the F plasmid, recipient bacterium including those from the bacterial chromosome, o As of 2014 about 80 species of bacteria may be transferred to the F− recipient cell were known to be capable of produces partial diploids, or merozygotes, transformation which are cells with two copies of some genes, Competence one on the bacterial chromosome and one on For transformation to take place, the recipient the newly introduced F plasmid. bacterium must be in a state of competence a temporary state of being able to take up exogenous DNA from the environment influenced by growth stage, the concentration of available DNA in the environment, and other environmental factors Natural competence o first step of transformation can occur Some species of bacteria take up DNA more without the second step, resulting in easily than others the addition of foreign DNA to the may occur naturally when dead bacteria break bacterial cytoplasm but not to its down and release DNA fragments into the chromosome environment Entry of DNA is thought to occur at a limited In soil and marine environments, transformation number of receptor sites on the surface of a may be an important route of genetic exchange competent bacterial cell for some bacteria o Passage into the cell is thought to be an active process that requires energy and Induced competence specific transport molecules increasing the frequency of transformation in Soon after entry, one of the two strands of the the laboratory in order to introduce particular double helix is digested by nucleases, leaving DNA fragments or whole plasmids into cells only a single strand to participate in o developed strains of bacteria that are transformation. The surviving DNA strand aligns more competent than wild-type cells with the complementary region of the bacterial o Treatment with calcium chloride, heat chromosome. In a process involving several shock, or an electrical field makes enzymes, this segment of DNA replaces its bacterial membranes more porous and counterpart in the chromosome, which is permeable to DNA excised and degraded. o efficiency of transformation can also be Once this is integrated into the chromosome, increased by using high concentrations the recombinant region contains one host of DNA strand (present originally) and one mutant strand. Because these strands are from different sources, the region is referred to as a heteroduplex, which usually contains some mismatch of base sequence. This mismatch activates a repair process. Following repair and one round of DNA replication, one chromosome is restored to its original DNA sequence, identical to that of the original recipient cell, and the other contains the properly aligned mutant gene. Following cell entry into cell is an active process (requires division, one non-transformed cell (nonmutant) energy and transport molecules) and one transformed cell (mutant) are pr foreign DNA can be added to the bacterial cytoplasm instead of the chromosome = NO recombination C. TRANSDUCTION recombinant region is called a heteroduplex o one host strand (original) + one mutant bacteriophages carry DNA from one bacterium strand to another o contains some mismatch of base o viruses that have bacteria as their hosts sequence viral DNA is an episome (just like F factor) o can replicate in the cytoplasm of a cell STEPS or PROCESS or as part of its chromosome (1) entry of foreign DNA into a recipient cell Head (2) recombination between the foreign DNA and its DNA can encode more than 150 genes homologous region in the recipient chromosome Base plate Collar = outer contractile sheath that surrounds coordinates the host cell recognition an inner spike-like tube provides signal whereby the outer sheath Base plate = from which tail fibers protrude contracts ➜ propel inner tube across host cell o consisting of 15 different proteins, most membrane present in multiple copies o coordinates the host cell recognition and is involved in providing the signal whereby the outer sheath contracts, propelling the inner tube across the cell membrane of the host cell Viral Infection period of intensive viral gene activity phage DNA replication occurs ➜ pool of viral DNA molecules synthesis of the viral head, tail, and tail fibers Bacteriophages have 2 alternative life cycles: 1. Lytic Cycle 2. Lysogenic Cycle Bacterial viruses (bacteriophages) carry DNA from one bacterium to another o Some viruses have DNA as their genetic material, whereas others have RNA; the nucleic acid may be double stranded or single stranded, linear or circular. INFECTION Viral DNA is classified as an episome (just like F factor) A period of intensive viral gene activity phage o a genetic molecule that can replicate DNA replication occurs, leading to a pool of viral either in the cytoplasm of a cell or as DNA molecules part of its chromosome the components of the head, tail, and tail fibers are synthesized Phage T4 1. DNA packaging as the viral heads are Head = has DNA contained within an assembled icosahedral protein coat (DNA can encode more 2. tail assembly than 150 average-sized genes) 3. tail-fiber assembly Tail = collar, tube, sheath  have 2 alternative life cycles: the lytic cycle and General Transduction Specialized Transduction the lysogenic cycle. phages integrate phages use specific randomly integration sites any gene may be only few genes transferred from near special sites one bacterial cell on the bacterial to another chromosome are transferred Recipient bacterium requires lysogenic receives only bacterial phages DNA (random) from donor bacterium. Recipient bacterium receives both bacterial DNA (adjacent to the integrated prophage) and viral DNA from donor bacterium. LYSIS / LYTIC CYCLE When approximately 200 new viruses are constructed, the bacterial cell is ruptured by the action of lysozyme (a phage gene product) the mature phages are released from the host cell The 200 new phages infect other available bacterial cells Lysogenic Cycle viral DNA (prophage) integrates into bacterial chromosome instead of replicating in the GENERALIZED TRANSDUCTION cytoplasm o phages integrate randomly viral DNA passed on to daughter bacterial cells o any gene may be transferred from one NO new viruses created bacterial cell to another by a virus NO lysis of bacterial cell in the lytic cycle of phage reproduction, the phage degrades the bacterial chromosome into Certain stimuli can trigger the lytic cycle again random fragments  In some types of bacteriophages, a piece of the 4 MAJOR INNOVATIONS IN MOLECULAR GENETICS bacterial chromosome, instead of phage DNA, occasionally gets packaged into a phage coat 1. Combination of DNA from different sources If a transducing phage infects a new cell and 2. Quick amplification of very small quantities of releases the bacterial DNA, the introduced specific DNA fragments genes may then become integrated into the 3. Development of quick and accurate methods of bacterial chromosome by a double crossover determining DNA sequences 4. Accurate and efficient editing of genome sequences Not all phages are capable of transduction. 4 major innovations in molecular genetics Requirements: allowed DNA from different sources to be Fragmentation of the bacterial chromosome. combined Non-specific packaging of DNA during phage allowed very small quantities of specific DNA assembly. fragments to be quickly amplified Recombination of the transferred bacterial development of quick and accurate methods of genes with the recipient's chromosome. determining DNA sequences accurate and efficient editing of genome sequences RECOMBINANT DNA TECHNOLOGY (GENETIC ENGINEERING) Not all phages are capable of transduction, a rare event that requires (1) that the phage degrade the bacterial chromosome (2) that the process of packaging DNA into the phage coat not be specific for phage DNA Recombinant DNA = DNA created by combining (3) that the bacterial genes transferred by the virus two or more fragments from different sources recombine with the chromosome in the recipient cell. DNA molecules from all organisms share the same chemical structure, differing only in the nucleotide sequence. II. DNA TECHNOLOGY The DNA sequences used can originate from any species. MOLECULAR GENETIC TECHNIQUES DNA sequences not found in nature can be Locate, analyze, alter, sequence, study, and chemically synthesized. recombine DNA sequences Probe the structure and function of genes Create commercial products (e.g. drugs, Recombinant DNA is the general name for a hormones, enzymes, and crops) piece of DNA that has been created by Diagnose and treat genetic diseases, hereditary combining two or more fragments from disorders and infectious diseases different sources. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure, differing only in the nucleotide sequence.  The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. o For example, plant DNA can be joined to bacterial DNA, or human DNA can be joined with fungal DNA. o DNA sequences that do not occur anywhere in nature can be created by the chemical synthesis of DNA and incorporated into recombinant DNA molecules. Using recombinant DNA technology and synthetic DNA, any DNA sequence can be created and introduced into living organisms recognize specific nucleotide sequences in DNA and make double-stranded cuts at those A. CUTTING & VISUALIZING DNA SEQUENCES sequences (called restriction sites) produced naturally by bacteria to defend 1. RESTRICTION ENZYMES (ENDONUCLEASES) against viruses recognize specific nucleotide sequences in DNA More than 800 different restriction enzymes and make double-stranded cuts that recognize and cut DNA at more than 100 produced naturally by bacteria to defend different sequences have been isolated from against viruses bacteria. Recognition Sequences Recognition sequences = sequences recognized by usually 4-8 bp long restriction enzymes located randomly within the genome usually 4-8 bp long used when DNA fragments must be cut or located randomly within the genome joined used when DNA fragments must be cut or most are palindromic joined most are palindromic = sequences that read the 2 types of fragment ends produced: same (5′ to 3′) on the two complementary DNA (1) Cohesive / sticky ends strands (2) Blunt ends 2 types of fragment ends produced 1. Cohesive / Sticky ends o fragments with short, single-stranded overhanging ends due to staggered cuts in the DNA 2. Blunt ends o by REs that cut in the middle of the recognition sequence o the cuts on the two strands are directly opposite each other  Engineered Nucleases complex enzymes designed to recognize longer DNA sequences limitation of restriction enzymes = their recognition sequences are short, typically 4–8 bp in length, they occur at random, many times within a genome e.g. over 47,000 BamHI restriction sites in the human genome. Cutting human DNA with BamHI or another restriction enzyme results in thousands of fragments. ❖ Thus, it is impossible to precisely cut genomic DNA at a single location with restriction enzymes. two cohesive/sticky end fragments that are cleaved by the same enzyme are consist of the part of a restriction enzyme that complementary to each other and can cleaves DNA nonspecifically, coupled with spontaneously pair or glue together another protein that recognizes and binds to a joined together permanently by DNA ligase, specific DNA sequence which seals nicks between the sugar–phosphate the particular sequence to which the protein groups of the fragments binds is determined by the protein’s amino acid sequence. 2. ENGINEERED NUCLEASES By altering the amino acid sequence of the complex enzymes designed to recognize longer binding protein, geneticists can custom-design DNA sequences a nuclease to bind to and cut any particular DNA sequence. Limitations of restriction enzymes (REs): o recognition sequences are short and occur at random, many times within a genome 3. CRISPR-CAS GENOME EDITING o cannot precisely cut at a single location for precisely cutting DNA o produce thousands of fragments CRISPR-Cas systems occur naturally in bacteria and archaea consist of the part of an RE (nuclease) that o to protect against foreign invaders cleaves DNA nonspecifically + another protein (bacteriophages, plasmids) that recognizes and binds to a specific DNA CRISPR Array sequence when foreign DNA enters prokaryotic cell, o specific sequence is determined by the proteins cut it up and insert bits of it into a protein’s amino acid sequence CRISPR array, (memory of the invader) has a series of repeating sequences (like a barcode) interspersed with the foreign DNA pieces (spacers) transcribed into crRNAs o combine with Cas proteins to form CRISPR-Cas complex = recognizes and cuts the same foreign DNA that was previously encountered  Protospacer-adjacent Motif (PAM) sequence that occurs at numerous random places throughout most genome associates with the CRISPR-Cas9 complex ➜ Cas9 unwinds DNA ➜ pair with complementary sequence in the DNA ➜ Cas9 makes double- stranded cuts in the DNA CRISPR-Cas Genome Editing for precisely cutting DNA CRISPR-Cas Immunity in Bacteria and Archaea CRISPR-Cas systems occur naturally in bacteria and archaea used to protect these organisms against bacteriophages, plasmids, and other invading DNA elements plasmids, and other invading Scientists have taken this natural system and modified it DNA for precise genome editing. A popular version is CRISPR- Cas9, which comes from the bacterium Streptococcus When a virus or foreign DNA invades, the bacteria cut it pyogenes. up and store pieces of it in their DNA, called a CRISPR array. This array has a series of repeating sequences (like Normally, CRISPR-Cas9 uses two RNA molecules, but a barcode) interspersed with these foreign DNA pieces. scientists have combined them into a single guide RNA (sgRNA). This guide RNA directs the system to the If the same invader shows up again, the bacteria use exact DNA sequence that needs editing. the stored pieces (called crRNA) to recognize and destroy the invader. The guide RNA matches a specific DNA sequence, and a protein called Cas9 cuts the DNA at that spot. For this to work, the target DNA must have a short, CRISPR-Cas9 specific sequence called a PAM (Protospacer Adjacent engineered from bacterium Streptococcus Motif), which helps the system find the correct place to pyogenes cut. more specific than restriction enzymes uses two RNA molecules combined into a single By changing the guide RNA, scientists can target and guide RNA (sgRNA) edit almost any DNA sequence with high precision, o directs the system to the exact DNA making it a powerful tool for genetic research and sequence that needs editing therapy. 4. GEL ELECTROPHORESIS gel acts as a sieve = separates molecules based Small wells are made at one end of the gel, solutions of on size and electrical charge DNA fragments are placed in the wells (Figure 19.4a), after cutting, resulting fragments should and an electrical current is passed through the gel. be separated to verify that the DNA was altered in the expected fashion Because the phosphate group on each DNA nucleotide determine the approximate size of the carries a negative charge, the DNA fragments migrate unknown fragments toward the positive end of the gel. o by comparing the migration distance of the unknown fragments with the During this migration, the porous gel acts as a sieve, distance traveled by size standards separating the DNA fragments by size. If the bands match the predicted fragment sizes, it Small DNA fragments migrate more rapidly than means that the DNA was cut correctly. do large ones, so, over 1–2 hours, the fragments separate on the basis of their size. Typically, DNA fragments of known length (size standards) are placed in one of the wells. By comparing the migration distance of the unknown fragments with the distance traveled by the size standards, a researcher can determine the approximate size of the unknown fragments Before cutting or altering DNA, scientists predict the sizes of the resulting fragments based on known sequences or where enzymes should cut. o If the DNA was altered as expected, the bands will match the predicted fragment sizes. o If the DNA fragments are not the expected size or the pattern looks different, this indicates that the DNA was not cut or altered correctly. Gel Electrophoresis to separate and visualize DNA fragments After cutting, it is often necessary to separate Visualization the resulting fragments and to verify that the stain the gel with a dye specific for nucleic acids, DNA was altered in the expected fashion. such as ethidium bromide, which wedges itself standard technique for separating molecules on tightly (intercalates) between the bases of DNA the basis of their size and electrical charge  and fluoresces when exposed to UV light, producing brilliant bands on the gel 5. PROBE a DNA/RNA molecule used to locate desired DNA fragments in a large pool of DNA has a base sequence complementary to a sequence in the gene of interest Limitations of Gel Electrophoresis: o some cuts made by REs produce thousand of fragments of different sizes = appear as a continuous smear on the gel Process: Cut the DNA into fragments by using one or more restriction enzymes Separates the fragments with gel electrophoresis Separated fragments must be denatured and transferred to a permanent solid medium (such as a nitrocellulose or nylon membrane) Probe membrane is placed in a hybridization solution for locating desired DNA fragments in a large containing a labeled probe pool of DNA probe binds to (hybridizes with) any DNA often a DNA or RNA molecule fragments on the membrane that bear with a base sequence complementary to a complementary sequences sequence in the gene of interest membrane is then washed to remove any The bases on the probe will pair only with the unbound probe bases on a complementary sequence, so, if A biochemical method reveals the presence of suitably labeled, the probe can be used to locate the bound probe a specific gene or other DNA sequence. Techniques to transfer denatured (single-stranded) Limitations of gel electrophoresis fragments from a gel to a permanent solid medium: Genomic DNA cut by a restriction enzyme Southern blotting produces thousand of fragments of different Northern blotting sizes = appear as a continuous smear on the gel - Transfer RNA from gel to solid support researchers are interested in only a few of these Western blotting fragments, perhaps those carrying a specific - transfer of protein from a gel to a membrane gene. Techniques to transfer denatured fragments from a gel to a solid medium: Southern Blotting (for DNA) Northern Blotting (for RNA) Western Blotting (for proteins)  B. AMPLIFYING SPECIFIC DNA FRAGMENTS Advantage/s Disadvantage/s Uses the cell’s high- Entire process (DNA fidelity replication insertion to DNA machinery = copies DNA isolation) takes several with great accuracy days Labor-intensive; difficult to automate AMPLIFY = increase the quantity of a particular DNA segment for further analysis or use 2 basic approaches: 1. Replicating DNA within cells (in vivo) 2. Replicating DNA enzymatically outside of cells (in vitro) Gene Cloning (In vivo) a DNA fragment is inserted into a bacterial cell Amplifying Specific DNA Fragments and the cell is allowed to replicate the DNA each gene is a tiny fraction of the total cellular Each time the cell divides, one or more copies of DNA. the DNA fragment are passed on to each Because each gene is so rare, it must be isolated daughter cell. and amplified before it can be studied. Most bacterial cells divide rapidly, so within a short time (usually a few days), a large number 2 basic approaches of genetically identical cells are produced, each replicating the DNA within cells (in vivo) carrying one or more copies of the DNA replicating the DNA enzymatically outside of fragment. cells (in vitro) The cells are then lysed to release their DNA, and the desired fragment is isolated from the rest of the bacterial DNA. 1. GENE CLONING (IN VIVO) Advantage o because it uses the cell’s high-fidelity replication machinery, gene cloning typically copies DNA with great accuracy Disadvantage o time required: the process of inserting the DNA into bacteria, selecting and growing the bacterial cells that have incorporated it, and isolating the amplified DNA usually requires several days o labor-intensive, requiring a number of steps that are difficult to automate CLONING VECTOR a replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell e.g. Plasmid Vector commonly used for cloning DNA fragments in bacteria bacteria take up plasmid + DNA fragment via TRANSFORMATION (uptake from environment) PLASMID VECTORS commonly used as vectors for cloning DNA fragments in bacteria. contain origins of replication and are therefore Require a CLONING VECTOR able to replicate independently of the bacterial a stable, replicating DNA molecule to which a chromosome. foreign DNA fragment can be attached for introduction into a cell easiest method for inserting a DNA sequence into a plasmid vector: Has 3 important characteristics: cut the foreign DNA (containing the DNA (1) an origin of replication fragment of interest) and the plasmid with the ensures that the vector is replicated within the same restriction enzyme cell; When DNA and plasmids are then mixed (2) selectable markers together, some of the foreign DNA fragments enable any cells containing the vector to be will pair with the cut ends of the plasmids. selected or identified DNA ligase is used to seal the nicks in the sugar– (3) one or more unique restriction sites into which a phosphate backbone, creating a recombinant DNA fragment can be inserted plasmid that contains the foreign DNA fragment. restriction sites used for cloning must be unique if a vector is cut at multiple recognition sites, Once a DNA fragment of interest has been placed inside several pieces of DNA are generated, and a plasmid, the plasmid must be introduced into bacterial getting those pieces back together in the correct cells. order is possible, but extremely difficult. Achieved via TRANSFORMATION = bacterial cells take up DNA from the external environment Inside the cells, the plasmids replicate and multiply as the cells themselves multiply. 2. POLYMERASE CHAIN REACTION (IN VITRO) can be used with extremely small amounts of amplify DNA enzymatically in a test tube outside original DNA, even a single molecule of cells requires a DNA template (the fragment of Primers interest) from which a new DNA strand can be short fragments of DNA, copied typically 17–25 nucleotides long complementary to known sequences on the template Solution that includes: the target DNA DNA polymerase all four deoxyribonucleoside triphosphates (dNTPs—the substrates for DNA polymerase) Primers magnesium ions and other salts (1) Denaturation (94-95 °C) (2) Annealing (50-56 °C) (3) Extension (72 °C) Primers short fragments of DNA complementary to known sequences on the template DNA Polymerase synthesizes new DNA strands Deoxyribonucleoside triphosphates (dNTPs) expand the growing DNA strand Magnesium ions and salts enhances the enzymatic activity of DNA 3 STEPS polymerase 1. a starting solution of DNA is heated to 90°– 100°C to break the hydrogen bonds between amplify DNA enzymatically in a test tube outside the strands and thus produce the necessary of cells single-stranded templates. The reaction mixture Because a DNA molecule consists of two is held at this temperature for only a minute or nucleotide strands, each of which can serve as a two template 2. DNA solution is cooled quickly to 30°–65°C and held at this temperature for a minute or less. Advantage During this short interval, the DNA strands do not have a chance to reanneal, but the primers allows DNA fragments to be amplified a are able to attach to the template strands. billionfold within just a few hours 3. the solution is heated for a minute or less to With each cycle, the amount of target DNA 72°C, the temperature at which DNA doubles, so the amount of target DNA increases polymerase can synthesize new DNA strands. At geometrically the end of the cycle, two new double-stranded  DNA molecules are produced for each original molecule of DNA. C. FINDING GENES OF INTEREST To analyze a gene or to transfer it to another organism, the gene must be located and isolated. Today, most genes are located by sequencing a genome and determining the locations of genes from the sequence. 1. DNA LIBRARY a collection of DNA fragments from an organism’s entire genome that are stored in a set of bacterial or viral hosts later on screened for genes of interest Creating a Genomic Library “SHOTGUN CLONING” = clone first, search later cells are collected and lysed, which causes them to release their DNA and other cellular contents Genomic library must contain a large number of into an aqueous solution, and the DNA is bacterial or phage clones to ensure that all DNA extracted from the solution sequences in the genome are represented in the library. After the DNA has been isolated, it is incubated with a restriction enzyme for a limited amount of time so that only some of the restriction sites in each DNA molecule are cut (a partial digestion). Because the cutting of sites is random, different DNA molecules will be cut in different places, and a set of overlapping fragments will be produced. The fragments are then joined to vectors and transferred to bacteria. A few of the clones contain the entire gene of interest (if the gene is not too large), and a few contain parts of the gene, but most contain fragments that have no part of the gene of interest. Screening the DNA Library to find target gene with the use of PROBES labeled with a fluorescent or radioactive marker grow bacteria or phages from the library on a petri dish o each colony or plaque contains a unique fragment of the genome Only the colonies or plaques that contain the DNA fragment with the gene of interest will bind the probe, and these will fluoresce. 1. IN SITU HYBRIDIZATION DNA is visualized while it is in the cell (in situ) Those clones can then be isolated, expanded, and determine the chromosomal or cellular analyzed location of a gene or its product o finding where a gene is expressed often helps define its function cells are fixed and the chromosomes be spread on a microscope slide and denatured Once a DNA library is created, it can be screened to find a gene or sequence of interest. The first step in screening is to plate (grow) the bacterial In Situ Hybridization cells or phages (viruses that infect bacteria) that contain DNA probes can be used to determine the the DNA fragments. chromosomal location of a gene Now you have a collection of colonies (or DNA (or RNA) is visualized while it is in the cell plaques) spread across a petri dish, each of (in situ). which contains a different piece of the genome. requires that the cells be fixed and the The goal is to find the colony or plaque that chromosomes be spread on a microscope slide contains the gene or DNA sequence of interest. and denatured. This is usually done by using a probe—a short sequence Determining where a gene is expressed often helps of DNA or RNA that is complementary to part of the define its function. gene they're looking for. For example, finding that a gene is highly expressed only in brain tissue might suggest The probe is labeled (often with a fluorescent or that the gene has a role in neural function. radioactive marker) and allowed to hybridize (bind) to the DNA from the colonies or plaques. Fluorescence in situ hybridization (FISH) denatured probes carry attached fluorescent dyes that can Only the colonies or plaques that contain the DNA be seen directly with the microscope fragment with the gene of interest will bind the probe, and these will light up under the right detection conditions, identifying the location of the gene. fluorescence in situ hybridization (FISH), the probes carry attached fluorescent dyes that can be seen directly with the microscope Several probes attached to different colored dyes can be used simultaneously to investigate different sequences or chromosomes used to identify the chromosomal location of human genes D. DETERMINING & ANALYZING DNA SEQUENCES a method for determining the nucleotide sequence of DNA. DNA Sequencing determines the sequence of can be performed manually or, more commonly, bases in a DNA molecule. in an automated fashion via sequencing It provides an enormous amount of information machine about gene structure and function. STEPS: DNA Sequence For Chain Termination PCR 1. DIDEOXY (SANGER) SEQUENCING DNA sequence of interest is used as a template for a special type of PCR called chain-termination PCR Step 1: DNA Sequence For Chain Termination PCR the addition of modified nucleotides (dNTPs) The DNA sequence of interest is used as a called dideoxyribonucleotides (ddNTPs). template for this special type of PCR the user mixes a low ratio of chain-terminating ddNTPs (dideoxyribonucleotides) are mixed ddNTPs in with the normal dNTPs in the PCR with regular dNTPs reaction. o ddNTPs lack a 3'-OH group, stopping ddNTPs lack the 3'-OH group required for DNA extension when incorporated phosphodiester bond formation; therefore, produces many DNA fragments of varying when DNA polymerase incorporates a ddNTP at lengths, each ending with a ddNTP, allowing for random, extension ceases. The result of chain- sequencing. termination PCR is millions to billions of oligonucleotide copies of the DNA sequence of interest, terminated at a random lengths (n) by 5’-ddNTPs. Step 2: Size Separation by Gel Electrophoresis Manual Sequencing the chain-terminated oligonucleotides are Read gel bands from bottom to top, checking separated by size which ddNTP (A, T, G, C) is associated with each the oligonucleotides will be arranged from band smallest to largest, reading the gel from bottom to top. Automated Sequencing o Shorter fragments end closer to the 5' a computer analyzes the fluorescent tags on the end of the original DNA sequence. gel Manual Sequencing the oligonucleotides from each of the four PCR reactions are run in four separate lanes of a gel to know which oligonucleotides correspond to each ddNTP reading the gel to determine the sequence of the input DNA. Because DNA polymerase only synthesizes DNA in the 5’ to 3’ direction starting at a provided primer, each terminal ddNTP will correspond to a specific nucleotide 1. Size Separation by Gel Electrophoresis in the original sequence the chain-terminated oligonucleotides are separated by size via gel electrophoresis. by reading the gel bands from smallest to largest, we In result, the oligonucleotides will be arranged can determine the 5’ to 3’ sequence of the original DNA from smallest to largest, reading the gel from strand. bottom to top. In manual Sanger sequencing, the In manual Sanger sequencing, the user reads all four oligonucleotides from each of the four PCR lanes of the gel at once, moving bottom to top, using reactions are run in four separate lanes of a gel. the lane to determine the identity of the terminal This allows the user to know which ddNTP for each band. For example, if the bottom band is oligonucleotides correspond to each ddNTP. found in the column corresponding to ddGTP, then the smallest PCR fragment terminates with ddGTP, and the first nucleotide from the 5’ end of the original sequence Step 3: Gel Analysis has a guanine (G) base. reading the gel to determine the sequence of the input DNA In automated Sanger sequencing, a computer reads the length of DNA fragments on the gel each band of the capillary gel, in order, using corresponds to the position where the ddNTP fluorescence to call the identity of each terminal ddNTP. terminated the chain. In short, a laser excites the fluorescent tags in each band, and a computer detects the resulting light (1) target DNA is cut into many short fragments emitted. (2) fragments are attached to a slide and amplified into clusters of ~1,000 copies each 2. NEXT-GENERATION SEQUENCING (3) primer binds to each fragment, and a sequences hundreds of times faster and less nucleotide is added expensive (4) a laser excites the fluorescent tag, revealing o millions of DNA fragments can be the nucleotide's identity by its color sequenced simultaneously (5) terminator and fluorescent are chemically removed Illumina Sequencing *entire process repeats* uses special nucleotides with different-colored fluorescent tags for A, T, C, and G each nucleotide has a reversible terminator = prevents addition of further nucleotides First, the DNA is cut into many short fragments, which Next-Generation Sequencing Technologies are attached to a slide and amplified into clusters. A sequencing hundreds of times faster and less primer binds to each fragment, and a nucleotide is expensive added. A laser excites the fluorescent tag, revealing the Most do sequencing in parallel, which means nucleotide's identity by its color. The stopper and tag that hundreds of thousands or even millions of are removed, and the process repeats. DNA fragments can be sequenced simultaneously, allowing, for example, a human By reading the flashes of light from each DNA cluster, genome to be sequenced in days instead of the sequence is determined. This method allows large years. amounts of DNA to be sequenced at once by reading many clusters simultaneously. E.g. Illumina Sequencing similar in principle to that used in dideoxy sequencing. E. ANALYZING GENE FUNCTION Special nucleotides are used that have a fluorescent tag attached, with a different 1. FORWARD & REVERSE GENETICS colored tag for each type of nucleotide. Each nucleotide also has a chemical group (a Forward Genetics Forward Genetics terminator) that, once incorporated into the growing DNA chain, prevents the incorporation Starts with a phenotype Starts with a genotype of any additional nucleotides. (observable traits) and (specific gene or DNA the terminator is reversible—it can be then identifies the gene sequence) and then chemically removed. responsible for it. understands the resulting phenotype.  (1) identify mutant (1) have a gene of 2. CREATING RANDOM MUTATIONS organism with unknown Before, geneticists relied on naturally occurring specific trait function mutations = rare and needs many organisms to (2) map the location (2) induce detect of the mutation mutations, alter on the genome sequence, or MUTAGENS (3) isolate and inhibit expression environmental factors that increase the rate of sequence the (3) observe effects mutation implicated genes on phenotype of organism increases the number of mutants in experimental populations of organisms MUTAGENESIS SCREENING To identify all genes affecting a phenotype, it’s ideal to create mutations in as many genes as possible Forward Genetics Starts with a phenotype (observable traits or mutations) and works toward identifying the gene responsible for that phenotype. Steps: identify mutant organisms with specific traits (e.g. hereditary defects) map the mutations causing the trait/problem Early in the study of genetics geneticists were the implicated genes could be isolated and forced to rely on naturally occurring mutations, sequenced which are usually rare and can be detected only if large numbers of organisms are examined. Reverse Genetics Starts with a genotype (specific gene or DNA The discovery of MUTAGENS—environmental sequence) and works toward understanding the factors that increase the rate of mutation— resulting phenotype provided a means of increasing the number of By altering the sequence or inhibiting its mutants in experimental populations of expression organisms. Begin with a gene of unknown function, induce o Radiation mutations in it, and then observe the effect of o Chemical mutagens these mutations on the phenotype of the o Transposable elements organism. depends on the ability to create mutations, not Mutagenesis screening = To determine all genes at random, but in particular DNA sequences, that might affect a phenotype, it is desirable to and then to study the effects of these mutations create mutations in as many genes as possible— on the organism that is, to saturate the genome with mutations 3. TARGETED MUTAGENESIS The new sequence with the mutation is Mutations are induced at specific locations replaced directly at the cut site. 2 STRATEGIES: 2. Oligonucleotide-directed Mutagenesis 1. Site-directed Mutagenesis if appropriate restriction sites are not available used in bacteria a single-stranded oligonucleotide is produced relies on using restriction enzymes to cut out a that differs from the target sequence by one or specific portion of the target DNA a few bases a synthetic oligonucleotide containing the the mismatch repair system in bacteria is key to desired mutation is inserted into the gap the success new sequence with the mutation is replaced directly at the cut site 2. Oligonucleotide-Directed Mutagenesis: Used when no suitable restriction sites are available for cutting the DNA. A single-stranded oligonucleotide (with the desired mutation) is introduced, which is very similar to the target DNA but contains a few Mutations are induced at specific locations base changes. 2 strategies This oligonucleotide binds to the target sequence through complementary base pairing, 1. Site-Directed Mutagenesis: despite the small mismatch in the base pairs. This technique relies on using restriction enzymes to cut out a specific portion of the The oligonucleotide then acts as a primer, target DNA. initiating DNA synthesis, which results in a A synthetic oligonucleotide containing the double-stranded DNA molecule with a desired mutation is inserted into the gap left by mismatch at the mutation site. the restriction enzyme cut.  When this DNA is inserted into bacteria, the An organism that has been permanently altered bacterial repair system will fix the mismatch. by the addition of a DNA sequence to its About half of the time, it will incorporate the genome is said to be transgenic, and the foreign mutation, while the other half will restore the DNA that it carries is called a transgene original sequence. TRANSGENIC MICE 4. TRANSGENIC ANIMALS often used in the study of the function of inserting DNA sequences into an organism's human genes genome (that normally lack such sequences) more similar to humans than are fruit flies, fish, observing effects of new sequences on and other model genetic organisms phenotype oocytes of mice and other mammals are large o foreign DNA = “transgene” enough that DNA can be injected into them directly. TRANSGENIC MICE DNA is injected into one of the pronuclei of mice are more similar to humans than other fertilized eggs using fine needles. model organisms Cloned DNA integrates randomly into egg cells (oocytes) are large enough to directly chromosomes via nonhomologous inject DNA recombination. o new DNA integrates randomly into the Embryos are implanted into a pseudopregnant chromosomes of the mouse via surrogate nonhomologous recombination foreign DNA ends up in all the cells of the mouse, including its reproductive cells About 10%-30% of embryos survive; a few carry the foreign DNA. because the DNA was injected at the one-cell stage of the embryo, these mice usually carry the cloned DNA in every cell of their bodies, including their reproductive cells, and will therefore pass the foreign DNA on to their progeny. adding DNA sequences of interest to the Through interbreeding, a strain of mice that genome of an organism that normally lacks such carry the foreign gene can be created. sequences and then observing the effect of the introduced sequences on the organism’s phenotype