Recombinant DNA Technology PDF

Recombinant DNA Technology Dr Thami Chiliza What is recombinant DNA?  Recombinant DNA Recombinant DNA technology is one of the recent advances in biotechnology The way in which genetic material from one organism is artificially introduced into the genom...

Recombinant DNA Technology Dr Thami Chiliza What is recombinant DNA?  Recombinant DNA Recombinant DNA technology is one of the recent advances in biotechnology The way in which genetic material from one organism is artificially introduced into the genome of another organism and then replicated and expressed by that other organism Was developed largely through the work of Herbert W. Boyer, Stanley N. Cohen, and Paul Berg in 1973 many other scientists made important contributions. Stanley N. Cohen , who received the Nobel Prize in Medicine in 1986 for his work on discoveries of growth factors. Stanley N. Cohen (1935–) (top) and Herbert Boyer (1936–) (bottom), who constructed the first recombinant DNA using bacterial DNA and plasmids.  What is Recombinant DNA Technology? RDNA technology - allows DNA to be produced via artificial means.  used to change DNA in living organisms & may have even more practical uses in the future.  It is an area of medical science that is just beginning to be researched in a concerted effort. RDNA technology works by taking DNA from two different sources  combining that DNA into a single molecule.  becomes useful when that artificially-created DNA is reproduced = DNA cloning.  Overview of Recombinant DNA Technology DNA containing gene of interest from a Vector, such as a different species is cleaved by an enzyme bacterium plasmid, is isolated. into fragments. Plasmid DNA containing gene of interest Bacterial cromosome recombinant DNA Desired gene is selected and inserted into plasmid. (plasmid) Plasmid is taken up by a cell, such as a bacterium. transformed bacterium Cells with gene of interest are cloned Create and harvest with either of two goals in mind. copies of a gene. Create and harvest or protein products of a gene. Plasmid RNA Protein product Gene encoding protein Gene encoding Amylase, cellulase, and Human growth hormone for pest resistance is degradative enzyme to other enzymes prepare treats stunted growth. inserted into plant cells. clean up toxic waste is fabrics for clothing inserted into bacterial manufacture. cells. Figure 9.1 A Typical Genetic Modification Procedure.  Brief History http://www.utsouthwestern.edu/edumedia/edufiles/education_training/programs/stars/holland-dnatech-history.pdf  Craig Venter—The Human Genome Project T en years ago, Craig Venter sequenced the first complete individual human genome - his own. Now, he's finally starting to decode what it means for his future. In early 2006, Craig Venter received a worrying email concerning his genome. Amid the six billion letters-long sequence of the A, T, C and G base pairs that constitute the vocabulary of DNA, geneticists at his private research institute had discovered a single, errant "C". This mutation, called APOE 4, marked him for increased risk of cardiovascular disease, and a tripled likelihood of Alzheimer's. The higher cardiovascular risk came as little surprise for someone with a family history of early heart-attack deaths. But the Alzheimer's was unexpected. http://www.wired.co.uk/article/craig-venter-human-longevity-genome-diseases-ageing In addition to having an entire institute's worth of geneticists on call, Venter is an undisputed pioneer in the field after leading his former company, Celera Genomics, in a fierce competition with the publicly funded Human Genome Project to produce the first complete human genome sequence. On June 26, 2000, in an uneasy truce that involved the intervention of then president Bill Clinton, the race was called as a tie.  Recombinant DNA Technology 1. The basic concepts for recombinant DNA technology 2. The basic procedures of recombinant DNA technology 3. Application of recombinant DNA technology  The basic concepts for recombinant DNA technology In the early 1970s, technologies for the laboratory manipulation of nucleic acids emerged. In turn, these technologies led to the construction of DNA molecules composed of nucleotide sequences taken from different sources. The products of these innovations, recombinant DNA molecules, opened exciting new avenues of investigation in molecular biology and genetics, and a new field was born— recombinant DNA technology.  Concept of Recombinant DNA RDNA is a molecule that combines DNA from two sources = gene cloning. Creates a new combination of genetic material Human gene for insulin was placed in bacteria The bacteria are recombinant organisms and produce insulin in large quantities for diabetics Genetically engineered drug in 1986 Genetically modified organisms are possible because of the universal nature of the genetic code!  Six steps of Recombinant DNA 1. Isolating (vector and target gene) 2. Cutting (Cleavage) 3. Joining (Ligation) 4. Transforming 5. Cloning 6. Selecting (Screening) What are plasmids?  Plasmids Plasmids – small circular pieces of DNA found primarily in bacteria  extrachromosomal DNA because they are in the cytoplasm in addition to the bacteria chromosome  Are small approximately 1 to 4 kb Found in species from the three representatives of the living world, namely, the domains Archaea, Bacteria, and Eukarya They can incorporate and deliver genes by recombination or transposition,  Favour genetic exchanges in bacterial populations  Plasmids…… Can replicate independently of chromosome Can be used as vectors – pieces of DNA that can accept, carry, and replicate other pieces of DNA Plasmids can be introduced into new hosts by a variety of mechanisms Found application in clinical, biotechnological & environmental © 2013 Pearson Education, Inc. Table 1.1. Examples of plasmids of E. coli Plasmid Size (Kb) Copy number Phenotype Conjugative ColE1 6.6 10 – 20 Colicin production and immunity No F 95 1-2 E. coli sex factor (pilus) Yes R100 89 1–2 Antibiotic resistance genes Yes P1 90 1–2 Present in prophages, produces viral No particles R6K 40 10 – 20 Antibiotic resistance genes Yes A color-enhanced electron micrograph of circular plasmid molecules isolated from E. coli A diagram of a typical DNA cloning plasmid. © 2012 Pearson Education, Inc. Figure 20.3  Plasmids are Classified by: 1. ABILITY TO BE TRANSFERRED TO OTHER BACTERIA Conjugative: sexual transfer of plasmids to another bacterium through a pilus. Non-conjugative: don’t initiate conjugation, transferred with the help of conjugative plasmids. 2. FUNCTION Fertility-(F) plasmids: are capable of conjugation (they contains the genes for the pili). Resistance-(R) plasmids: contain gene(s) for resistance against one/more antibiotics/poisons. Col-plasmids: contain genes coding for colicines - proteins that can kill other bacteria. Degradative plasmids: able to digest unusual substances, e.g., toluene or salicylic acid. Virulence plasmids: turn a bacterium into a pathogen. 3. COPY NUMBER High copy number = 10-100 copies/cell Low copy number = 1-4 copies/cell  Plasmid DNA Replication Most E. coli plasmids use DNA polymerase III as central replication enzyme  polymerase synthesizes leading and lagging strand DNA in prokaryotes.  An important exception is ColE1 plasmid family  In which DNA polymerase I plays the key replication role Rep protein, it plays a key role in initiation of plasmid replication.  Plasmid copy number is related to intracellular level of Rep protein.  Function of Rep protein is to recognize specific short DNA sequences at plasmid ori Replisome  Plasmid as Vectors Vectors - small pieces of DNA used for cloning (the gene to be inserted into the genetically modified organism must be combined with other genetic elements in order for it to work properly) Requirements of the Vector 1. Self-replication - able to replicate in the host (origin of replication) 2. Cloning site (site for recognition of restriction nucleases) 3. Promoter (and operator) - to support the gene (new DNA) expression in the host 4. Selectable marker – antibiotic resistance 5. Proper size © 2012 Pearson Education, Inc. Figure 20.4  Six steps of Recombinant DNA 1. Isolating (vector and target gene) 2. Cutting (Cleavage) 3. Joining (Ligation) 4. Transforming 5. Cloning 6. Selecting (Screening)  Recombinant DNA It is important to understand the key steps involved in cloning a gene.  DNA is cut into fragments and introduced into a new host, usually E. coli, where it is copied. We cannot simply introduce fragments of DNA into a cell or organism  they will probably be degraded, and  even if it is not it will not be replicated and passed on when the cell divides. To ensure that the piece of cloned DNA is copied and passed on  it is necessary to put it into a vector  ensure that it is copied every time that the cell copies its own DNA  a copy is passed on to each daughter cell at cell division Cloning involves cutting the vector and joining in the piece of DNA  This cutting and joining of DNA fragments is done using enzymes  The new DNA molecule that was created is introduced into your host cell  by a process called transformation = in the host it will be copied and passed on daughter cells Use of Restriction Enzymes & DNA Ligase to make recombinant DNA molecules Purified and characterized the cleavage site of a Restriction Enzyme.  What are restriction enzymes? Molecular scissors that cut double stranded DNA molecules at specific points.  Restriction Enzymes Restriction endonucleases cuts DNA at specific nucleotide sequences = restriction sites. REs are found in many different strains of bacteria where their biological role is to participate in cell defence.  Enzymes selectively cut up (restrict) foreign (e.g. viral) DNA that enters the cell, in a process called restriction.  prevent the replication of the phage by cleaving its DNA at specific sites. Host DNA is protected by Methylases which add methyl groups to A or C bases  modifying the site and protecting the DNAfrom cleavage. To cut the DNA, restriction enzyme makes two incisions, each strand of the DNA double helix. Biological role of REs  Types of Restriction Enzymes Use in Class Abundance Recognition site Composition recombinant DNA research Type I Less common than typeCut both strands at a Three-subunit complex: Not useful II nonspecific location > individual recognition, 1000 bp away from endonuclease, and recognition site methylase activities Type II Most common, Cut both strands at a Endonuclease and Very useful approximately 240 specific, usually methylase are separate, enzymes commercially palindromic, recognition single-subunit enzymes available site (4-8 bp) Type III Rare Cleavage of one strand Endonuclease and Not useful only, 24-26 bp methylase are separate downstream of the 3′ two-subunit complexes recognition site with one subunit in common  Restriction Enzymes scan the DNA sequence Find a very specific set of nucleotides Make a specific cut  Picking a palindrome Words that read the same forwards as backwards Hannah hannaH Level leveL Madam madaM  Palindromes in DNA sequences Genetic palindromes are similar to verbal palindromes. 5’ 3’ A palindromic sequence in DNA is one in which the 5’ to 3’ base pair sequence is identical on both strands. Restriction enzymes recognize and make a cut within specific palindromic sequences, known 3’ 5’ as restriction sites, in the DNA. This is usually a 4- or 6 base pair sequence.  Cleavage Patterns of Some Common Restriction Endonucleases Restriction fragments can be blunt ended or sticky ended © 2012 Pearson Education, Inc. Figure 20.1  Cohesive (‘sticky’) ends These are products of restriction enzyme digestion with protruding ends  these ends are known as cohesive, or ‘sticky’ ends  since they can bind to any other end with the same overhanging sequence,  by base pairing (annealing) of the single-stranded tails For example, any fragment formed by an EcoRI cut  can anneal to any other fragment formed in the same way, and  may subsequently be joined covalently by ligation In some cases, DNA ends formed by enzymes with different recognition sequences  may be compatible,  provided the single-stranded tails can base-pair together.  Restriction Enzymes Over 3000 have been identified More than 600 available commercially Routinely used for DNA modification and manipulation in laboratories. Isoschizomers?  Different enzymes identified from different bacteria may have the same recognition sequence, with the same cleavage site.  Some enzymes recognize similar (not same) sequences but produce the same sticky ends. http://en.wikipedia.org/wiki/Restriction_enzyme  Naming of restriction enzymes The names for restriction enzymes come from:  the type of bacteria in which the enzyme is found  the order in which the restriction enzyme was identified and isolated.  Nomenclature EcoRI E = Escherichia genus name co = coli species name R = strain RY12 strain or serotype I = Roman numeral one = first enzyme HinDIII Haemophilus influenza serotype d 3rd enzyme  Six steps of Recombinant DNA 1. Isolating (vector and target gene) 2. Cutting (Cleavage) 3. Joining (Ligation) 4. Transforming 5. Cloning 6. Selecting (Screening) DNA Ligase  DNA Ligase Now that we know how to cut DNA molecules  we need to consider how to join them together in a new combination.  the new molecule is called a recombinant DNA cut up using a restriction enzyme like EcoRI  produces sticky ends,  then when two molecules with the same sticky ends come into contact,  hydrogen bonding between the complementary bases will cause the molecules to stick together This is in fact why these molecules are said to have sticky ends.  this is not a very stable arrangement  the two molecules will soon drift apart again For gene cloning, you need to be able to covalently link the two molecules.  enzyme that is capable of doing this is called DNA ligase.  Formation of a covalent phosphodiester bond Two restriction fragments with sticky ends are held together by hydrogen bonding  are in effect two single-stranded breaks in a double-stranded molecule;  DNA ligase repairs these single-stranded breaks DNA ligase catalyzes the formation of a covalent phosphodiester bond  between the 5′ phosphate on one DNA strand and a 3′ hydroxyl on another  this process requires energy Most commonly used DNA ligase is a protein produced by a called T4  T4 DNA ligase  It uses ATP as an energy source ATP © 2012 Pearson Education, Inc. DNA Ligase Mode of Action repairs nicks in DNA strands reforms phosphodiester bond uses energy from ATP works on blunt or sticky ends Restriction enzymes + DNA ligase = Recombinant DNA Figure 8.2 Other enzymes  Phosphatases Ligation is used to covalently join passenger DNA to the vector DNA. However, the vector DNA can also self-anneal  be ligated without any foreign or passenger DNA inserts = not desirable A 10: 1 ratio of passenger DNA to vector DNA  increase the possibility of passenger – vector DNA annealing = being ligated Calf intestinal phosphatase used = remove 5′ phosphate of linearized vector DNA  vector DNA will not ligate to itself since the 5′ phosphate is required for this reaction Since the passenger DNA has 5′ phosphate, there will be some ligation due to the two 5′ phosphates provided passenger DNA. However no ligation will take place at the two other ligation points where the 5′ phosphates are provided by the vector DNA.  DNA polymerase I Synthesizes DNA complementary to a DNA template in a 5′ to 3′ direction beginning with a primer with a free 3′-OH. The Klenow fragment is a truncated version of DNA polymerase I which lacks the 5′ to 3′ exonuclease activity.  RNA dependant-DNA polymerase (reverse transcriptase) Reverse transcriptase catalyzes synthesis of a ssDNA from mRNA template Like DNA polymerase, reverse transcriptase (RT) also needs a primer to get started.  it can have 5′-3′ exoribonuclease activity and  3′-5′ exoribonuclease activity that specifically degrades RNA in DNA-RNA hybrid molecules. RT is purified from RNA tumour viruses = used to transcribe mRNA into dsDNA  First cDNA is synthesized and then the RNA is degraded by alkali or ribonuclease H.  Second strand synthesis is then carried out using Klenow fragment of DNA polymerase I or RT itself. In this synthesis cDNA acts as its primer and template through formation of a hairpin. It must be highlighted that RT it has no proofreading ability. © 2012 Pearson Education, Inc. Figure 20.6  Six steps of Recombinant DNA 1. Isolating (vector and target gene) 2. Cutting (Cleavage) 3. Joining (Ligation) 4. Transforming 5. Cloning 6. Selecting (Screening) Ways to get DNA into bacteria cells Plasmids, BACs & sometimes PACs Cosmids & sometimes PACs Lambda Recombinant DNAs  Bacterial Transformation Traditional method involves incubating bacterial cells in concentrated calcium salt solution The solution makes the cell membrane leaky, permeable to the plasmid DNA Newer method uses high voltage to drive the DNA into the cells in process called electroporation 4-59  Competent Cells E. coli cells are more likely to incorporate foreign DNA  if their cell walls are altered so that DNA can pass through more easily. Such cells are said to be "competent." Cells are made competent by a process that uses calcium chloride (CaCl2) and heat shock. Cells that are undergoing very rapid growth are made competent more easily than cells in other stages of growth. The actual length of each phase depends on the temperature at which the cells are incubated.  cells that should be in the log phase.  Screening Transformants Transformation produces bacteria with:  Religated plasmid  Religated insert  Recombinants Identify the recombinants using the antibiotic resistance  Grow cells with tetracycline so only cells with plasmid grow, not foreign DNA only  Next, grow copies of the original colonies with ampicillin which kills cells with plasmid including foreign DNA 4-62  Screening With Replica Plating Replica plating transfers clone copies from original tetracycline plate to a plate containing ampicillin A sterile velvet transfer tool can be used to transfer copies of the original colonies Desired colonies are those that do NOT grow on the new ampicillin plate 4-63  pUC and b-galactosidase Some plasmids such as pUC plasmids have: Ampicillin resistance gene Multiple cloning site inserted into the gene lacZ’ coding for the enzyme b- galactosidase  Clones with foreign DNA in the MCS disrupt the ability of the cells to make b- galactosidase  Plate on media with a b-galactosidase indicator (X-gal) and clones with intact b- galactosidase enzyme will produce blue colonies  Colorless colonies should contain the plasmid with foreign DNA 4-64  Directional Cloning Cut a plasmid with 2 restriction enzymes from the MCS Clone in a piece of foreign DNA with 1 sticky end recognizing each enzyme The insert DNA is placed into the vector in only 1 orientation Vector re-ligation is also prevented as the two restriction sites are incompatible 4-65 Double digests and directional cloning End 10 September CLONING VECTORS  Cloning vectors DNA molecules that can carry a foreign DNA segment into the host cell The vectors used in recombinant DNA technology can be plasmids, cosmids, bacteriophages etc. Plasmids: Self-replicating, circular, extra chromosomal DNA present in bacteria.  Plasmids have only one or two copies per cell. Bacteriophages: Virus infecting bacteria.  Bacteriophages have high number per cell, so their copy number is also high in genome Cosmids: Hybrid vectors derived from plasmids which contain cos site of lambda phage.  Plasmids Plasmid: Small circular DNA that can replicate independently from a host’s chromosome. Characteristics: Smaller than chromosome (