Lecture 5 (Recombinant DNA)

Agrobacterium-mediated transformation

(Step 1) Wounded plant cell produces signal molecules. (Step 2) Signal molecules are recognized by bacterial receptors. (Step 3) Agrobacterium attaches to plant cell. (Step 4) VirG activates transcription of vir genes, resulting in proteins for transport and T-DNA complex formation. (Step 5) T-DNA is excised from Ti plasmid to form immature T-complex. (Step 6) Immature T-complex is transported to the plant cell. (Step 7) T-complex matures by binding to VirE2 proteins and moves to the nucleus. (Step 8) T-DNA randomly integrates into a plant chromosome, and bacterial genes are expressed.

site-directed mutagenesis (SDM)

-A technique for examining the role of individual amino acid residues in proteins. -It involves the use of recombinant DNA techniques to selectively replace the residue of interest with a different amino acid with critically different properties. -The resulting protein can then be tested functionally, e.g. with respect to substrate binding or catalytic activity. Principle of site-directed mutagenesis. The single- stranded DNA could be that of the single-strand DNA phage M13 containing the cloned gene; alternatively, if the gene had been cloned in a double-stranded vector, the DNA would first need to be denatured, to allow hybridization with the synthetic oligonucleotide. Several rounds of replication of the ligated product introduced into a bacterial cell will result in the formation of a mutated sequence of the original DNA. Convenient methods exist to get rid of the latter.

blunt vs sticky ends

-Both kinds of products can be ligated to form uninterrupted molecules, but the ligation efficiency of blunt-ended fragments is much lower than that of fragments containing overhangs, because the latter can actually base-pair with each other, increasing the probability of the two ends staying in close proximity and in proper orientation for ligation to occur.

overall process of gene therapy

-Gene therapy is a complex multistep process involving several steps in the production of the vector carrying the gene of interest. -Once the vector delivers the transgene into the cell, the gene must travel through the cytoplasm and enter the nucleus, where it should be stably integrated into the nuclear genome: only integrated gene copies can be consistently replicated as part of the genome. -Finally, properly regulated expression must be achieved. This is not a trivial task, as the majority of vectors will insert the gene they carry at random locations. --> Such random insertion can create two kinds of problems. (1) In most cases, the gene will end up in a heterochromatic environment that does not permit its transcription. (2) In other cases, the gene may land within other genes or their regulatory sequences, which will lead to the inactivation of these host genes. This may be detrimental to the host cell, to an extent that depends on the importance of the inactivated gene.

DNA linkers

-Joining together blunt ends is much more challenging, and the enzymes that are capable of doing this are much less efficient. -The efficiency of blunt- end ligation can be increased by using small oligonucleotides that carry restriction sites for sticky-end production, known as DNA linkers. -Figure 5.8 DNA linkers for joining of blunt-end DNA fragments. Linkers can be chemically synthesized to contain any restriction target site. Blunt-end ligation is performed by the DNA ligase from phage T4, as the E. coli DNA ligase will not catalyze blunt-end ligation except under special reaction conditions of macromolecular crowding. Linkers can be ligated to both ends of the blunt-end DNA fragment to be cloned; upon treatment with the restriction endonuclease that recognizes the linker sequence, sticky ends are produced. This treatment is then followed by standard cloning techniques that use sticky ends for efficient ligation of the foreign gene into a vector treated with the same enzyme.

restrictases bound to cognate vs non cognate DNA

-Numerous restrictases have been crystallized and their structures have been determined in their free forms, bound to noncognate (nonspecific) DNA fragments, and bound to cognate sequences. -Figure 5.5 compares the crystal structures of EcoRV when bound to a random sequence and when bound to its specific recognition site. -Note that interaction with the cognate nucleotide sequence leads to conformational changes in the enzyme that allow it to embrace the DNA more tightly.

uses for recombinant bacteriophage

-Plasmid vectors are often used for cloning small DNA segments, up to 10 kbp. Clon- ing larger fragments, such as individual large genes or fragments to be used for the construction of genomic libraries, requires the use of alternative vectors, such as derivatives of bacteriophages. -The most widely used phage for cloning is phage λ and its derivatives.

animal cloning

-Such clones can be produced by the technique of nuclear transfer. This method involves taking a cell nucleus from the donor and injecting it into an enucleated egg. -This is then fertilized and allowed to develop as an embryo, which is then transplanted into the uterus of a foster mother. -proved principle of genetic equivalence: all cells of an adult organism contain the same genetic information and differ only in what portion of that information is being used in a specific cell type. -Thus, nuclei from cells that are differentiated retain all the genetic information needed for the development of an entire organism; they just need to be reprogrammed, for example, by being transferred to the environment of an enucleated egg.

Type 2 restriction enzymes are the most useful restriction enzymes for biological applications because

-The most useful restriction enzymes for biological applications because the have the ability to recognize a particular DNA site and to cut at a defined point within that site -The cleavage patterns of type II restrictases can be rather diverse.

knockout mice

-The procedure begins with the construction of a vector containing the interrupted gene, together with enough native flanking sequences to favor homologous recombi- nation in the recipient cell -The vectors carrying the altered gene are inserted, by chemical means or by electroporation, into embryonic stem or ES cells taken from a mouse embryo (These are pluripotent cells that have the potential to develop into any kind of cell of the organism if provided the right molecular cues from the environment.) -The cells are selected for recombination events during growth in a culture medium. Recombinant cells are then inserted into another blastocyst mouse embryo -The resulting mixed embryo that contains both normal cells and cells with the interrupted gene is implanted into the uterus of a foster mouse. -The newborn mice will be chimeras, with a mosaic of altered heterozygous cells and wild-type cells. -If such females are allowed to grow to maturity and bred to wild-type males, some of the progeny will be wild-type while the rest will be heterozygous for the interrupted gene. -Modifications of the above procedure can be used to generate knock-in organisms, in which a modified gene is specifically substituted for the wild-type gene, or knock- downs, in which the regulation of a particular gene is modified.

ways to overcome the nonviral vectors' impaired ability to enter nucleus

-The simplest option is the direct introduction of therapeutic DNA into target cells. The use of liposomes, artificial lipid spheres with an aqueous core containing the DNA, is another option. -In still another approach, the DNA is chemically linked to molecules that are capable of binding to special cell-surface receptors or of facilitating nuclear transfer. -human artificial chromosomes, with practically unlimited capacity and stability of gene expression and no immunogenicity.

viral vector

-The viral vector must be replication-incompetent: that is, it must carry only the essential elements necessary for transgene packaging and expression. -The viral genes necessary for viral particle production are removed from the vector and provided in trans by another virus in the vector-producing cell lines.

Type 2 restriction enzymes usually recognize

-These enzymes usually recognize palindromic sequences, which read the same on both complementary DNA strands, 5′ → 3′ and 3′ → 5′, and bind as homodimers.

general characteristics of expression vectors

-They should be present and maintained in the host cells at high copy number. -They should be able to integrate into the host genome to ensure stable propa- gation of the gene of interest from cell generation to cell generation. -They should carry a promoter that is functional within the given host. Promot- ers are elements where RNA polymerases bind to initiate gene transcription (see Chapters 11 and 12). -It should be possible to regulate the promoter to ensure that the gene is expressed only when desired (see Chapters 11 and 12). This is especially important if the protein happens to be toxic to the host cell.

Type II restriction enzymes produce

-Type II restriction enzymes produce 5′-phosphates and 3′-OH groups only.

how do viruses enter the cell?

-Viruses enter the cell through endocytosis, which is triggered by binding of the virus to spe- cific receptor proteins on the cell surface. These may or may not be present on all cells. -Modifications of the viral genes that encode the envelope and capsid proteins can overcome this problem.

transgenic mice procedure

-a technique for introducing new genetic material into eukaryotic genomes in a way that would be transmitted from generation to generation. -an egg is removed from a female mouse and fertilized in vitro. -The vector containing the gene of interest, as well as sequences necessary for proper transcription of the gene and markers to allow screening for proper incorporation, is injected into the male pronucleus, the haploid nucleus of the sperm, after the sperm enters the egg. -The timing of the microinjection is critical because the introduced DNA must integrate into the genome prior to the duplication of genetic material that takes place before the first embryo cell division. -If integration occurs following that division, the mouse will be mosaic, with some wild-type and some transgenic cells. Integration of the vector into the host genome is random with respect to the underlying genomic DNA sequence. -At the next stage, the egg is transplanted to the uterus of a foster mouse, and a pup will be born. If integration is successful, the resulting mouse will be heterozygous in the region of integration. -However, it must be remembered that this is the addition of a function; the original variant of the gene is still present and, in most cases, functional. If we want to take away a gene or replace it with an altered version, the challenge is somewhat more difficult.

Recombinant DNA technology

-allowed us to mutate what we cloned and then study proteins with targeted changes on its sequence, which may change the properties of the proteins -allowed for biochemical characterization of pathways and physiological roles by introducing modified genes into living cells -can modify the genetic content of cells and whole organisms via genetic engineering

Replacement vectors

-allows approximately 15 kbp

how to clone large DNA fragment (yeast artificial chromosome or YAC)

-allows cloning of fragments larger than 100 kbp and up to 3000 kbp. -In addition to the cloning of large genes, YACs are useful for physical mapping of com- plex genomes and for constructing genomic libraries. -YACs and other yeast expression vectors have an advantage over BACs in that they can be used to express eukaryotic proteins that require post-translational modifications. -The disadvantage of YACs is that they are less stable than BACs, allowing significant gene rearrangements due to high copy numbers.

DNA ligases

-among the most important enzymes in the cell. They are indis- pensable for numerous cellular processes, including DNA replication, recombina- tion, and repair, whenever there is a need to seal nicks in the DNA double helix or join broken helices. -Of interest to us here is their essential role in joining frag- ments of double-helical DNA to form intact molecules in recombinant DNA tech- nologies. -Ligases form phosphodiester bonds between the 3′-OH and 5′-phosphate termini of DNA fragments -The reaction involves several steps. Ade- nylation of the active-site lysine uses ATP in phages and eukaryotes but uses NAD+, nicotinamide adenine dinucleotide, in bacteria.

EcoRV endonuclease

-complexes with DNA -no ATP required

isoschizomers

-different restriction enzymes that cleave the same DNA sequence -have the same recognition site but come from different bacterial species -they may cut the sequence at different places, thus adding to the diversity of cleavage patterns and DNA ends produced.

golden rice

-has been engineered to produce β-carotene (provitamin A) in its endosperm, the edible part of rice. Note that rice plants naturally produce β-carotene in the leaves but not in the endosperm. In addition, the plant was engineered to increase its iron content and to improve iron absorption.

nonviral vectors

-have been developed in order to overcome the immunogenicity and safety issues linked to the use of viral vectors. -Although nonviral vectors are much safer then viral vectors, they have an important limitation—an impaired ability to enter the nucleus—which has so far limited their use to proliferating cells only.

2 types of phage λ used for recombinant technology

-insertion vectors, the linear phage DNA molecule is cut with a restriction enzyme that cleaves the sequence only once. The two fragments generated are joined to the foreign DNA fragment, and the resulting recombinant is packaged into infectious mature viral particles -In order to overcome the strict limitation on the size of foreign DNA that can be cloned, replacement vectors. These vectors make use of the fact that about a third of the λ genome is not necessary for replication and can thus be replaced by any piece of foreign DNA without affecting the infectivity of the recombinant phage.

Methylation-sensitive isoschizomers

-may or may not cleave a sequence, depending on whether the sequence carries methyl groups. -These pairs of enzymes have proven instrumental in studying the methylation patterns of specific sequences of interest.

type 1 restrictases and its 3 subunits

-multifunctional enzymes that combine both nuclease and methylase activity in a single protein trimeric complex -One subunit, R, carries the restriction function, cleaving the DNA at random locations, -it also has an ATP-dependent motor activity that is involved in translocating the DNA. -The second subunit, M, carries out the DNA methylation reaction, which occurs in the rec- ognition site itself. -The third subunit, S, recognizes the specific sequence to which the complex binds. For cleavage to occur, the recognition/binding site has to be brought into proximity with the cleavage site; that is, the intervening DNA has to loop out (at the same time it is supercoiled) -Therefore, these enzymes are unusual molecular motors that bind specifically to DNA and then move the rest of the DNA through this bound complex.

restriction endonucleases

-recognize specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides -Each restriction enzyme is characterized mainly by the specific sequence it recognizes and the site where it cuts the double helix

gene therapy

-the transplantation of normal genes into cells in place of missing or defective ones in order to correct genetic disorders. -Gene therapy combines recombinant techniques with the aim of correcting defective genes or gene functions responsible for disease development. Administration of the functional gene, instead of the protein itself, is performed because proteins are quickly degraded, whereas a properly integrated gene will continue to be expressed.

Phage Display Technology

-utilizes bacteriophage M13 as a vector given that its coat is amazingly flexible, changing its size to accommodate DNA molecules of very different sizes, ranging from a few hundred nucleotides to around twice the size of the native genome. -This method allows large peptide and protein libraries to be expressed on the surface of the filamentous phage and thus presented to potential interaction partners. -It permits the selection of expressed peptides and proteins, including antibodies, that have high affinity and specificity for almost any target, including specific proteins or DNA sequences. Thus, its main use is for finding protein-protein and protein-DNA interactions. -The direct link between the experimental phenotype, that is, the properties of the peptides or proteins on the surface, and the genotype encapsulated within the vector allows the selection of molecules that are optimized for binding. -In an important application, phage display facilitates the engineering of antibodies to amend their size, affinity, and effector functions.

ligation in recombinant DNA

1)Attack of Lysine on the a-phosphate of ATP, release of inorganic pyrophosphate 2)Forms Enzyme-AMP complex 3)The 5' P of nicked DNA attacks the Lys-AMP intermediate forming a pyrophosphate linkage (App-DNA) 4) The 3'-OH end (nicked strand) attacks the 5'P of the App-DNA 5) This covalently joins the nicked DNA and releases AMP

2 gene therapy approaches

1. A normal gene may be inserted into a random, nonspecific location within the genome to supplement a nonfunctional gene. This approach is classified as gene addition therapy, since the nonfunctional gene stays in the genome. Examples might be the use of tumor suppressor genes in the treatment of a malignancy or immunostimulatory genes to treat an infectious disease. Gene addition therapy could also be used to provide the cell with transgenes whose expression can control the expression of a mutated gene. 2. A mutated gene can be swapped for a normal gene through homologous recombination. This approach is representative of gene replacement therapy.

2 approaches for sequencing large genomes

1. whole-genome shotgun approach 2. shotgun sequencing approach (also called BAC-based or clone-by-clone sequencing)

Ti plasmid (tumor inducing)

Agrobacterium tumefaciens bacteria infects certain plants, its plasmid that can be used to insert a foreign gene into a plant -the T-DNA region is the portion of the plasmid that is transferred to the plant cell and stably integrated into the plant nuclear genome. • If successful, plant has a tumor and the foreign gene -the native wild-type oncogenes and the opine synthase genes can be re- placed by any sequence of interest, which will then be trans- ferred to the plant cell and integrated into the genome.

HIV detection methods:

All three common methods for diagnosing HIV infection have been developed through DNA re combinant methods. Principles of the two immunological tests, ELISA or enzyme-linked immunosorbent assay and Western blots: they detect antibodies to specific recombinantly produced HIV proteins that are present in blood samples from patients. The test that directly detects the RNA genome of HIV uses reverse transcriptase polymerase chain reaction or RT-PCR. Development of the RT-PCR test was made possible by molecular cloning and sequence analysis of the HIV genome. Figure: (A) Enzyme-linked immunosorbent assay (ELISA) test. Blood samples from three patients were analyzed by ELISA. In the case of positive ELISA, a Western blot test is performed. (B) Western blot test. The interpretation of this test is not as straightforward as that of ELISA, as the viral protein bands that are detected by antibodies present in the blood sample are not always visible. The consensus among clinicians is that, to be conclusive, the profile must have at least five bands.

Insertion vectors

Figure 5.10 Packaging of λ phage DNA into infectious mature viral particles. The heterodimeric terminase enzyme complex and the E. coli integration host factor (IHF) bind cooperatively to a cossite in a DNA concatemer. This is the form of the viral genome that is produced during replication; it contains multimers of genome-size fragments connected by cos sequences. The terminase possessescos-site cleavage endonuclease, helicase, and ATPase activities that work in concert to assemble the packaging machinery on the concatemer and prepare the end for packaging. The same enzyme also possesses a DNA- and ATP-dependent translocase activitythat packages DNA into an empty procapsid, thus serving as a molecular motor. The procapsid is an empty protein shell composed of four different proteins; the tail is composed of 11 different viral glycoproteins. The terminase-concatemer complex, named the initiation complex, binds to the portal, a ringlike structure in the procapsid, to form the motor complex which pumps the viral DNA into the capsid. DNA packaging triggers a process of expandingthe procapsid, with additional viral proteins binding to it. Once the packaging motor encounters the next cos site in the concatemer, terminase again cuts the duplex, and its helicase activity helps in packaging the genome to near-liquid-crystalline density. Following the addition of some more proteins to prevent DNA release through the portal, the preassembled tail is added to complete virion assembly. It is now possible to assemble infectious viral particles in vitro starting with seven individual proteins, purified procapsids and tails, and mature λDNA. Importantly, the same system can be used to produce infectious viruses containing recombinant DNA.

steps of phage display method

Figure 5.13 Phage display identifies DNA sequences that encode peptides or proteins of desired characteristics. Asan example, we present the sequence of steps in phage display screening to identify polypeptides that bind with high affinity to desired target protein or DNA sequence. (Step 1) Target proteins or DNA sequences are immobilized to the wells of a plastic microtiter plate. (Step 2) A library of DNA sequences that code for peptides/ proteins is prepared by inserting these sequences into the gene encoding coat protein 3. Other coat proteins can also be used, including the major protein p8 (see Figure 5.12). Thus, the products expressed by the recombinant genes are fusion proteins between p3 and the peptide/protein encoded by the inserted DNA sequence. The fusion proteins end up on the phage surface. (Step 3) This phage-display library is added to the plate and allowed time to bind. Then a buffer wash removes the unbound phages, while those carrying the fusion proteins that interact with the immobilized target remain bound. (Step 4) The specifically attached phages are eluted with excess known ligand for the target or by lowering pH. The eluted phages are used to generate more phages by infection of suitable bacterial hosts. The new phage mixture is enriched, containing considerably fewer irrelevant (nonbinding) phages than were present in the initial mixture. (Step 5) Steps 1-4 are repeated to achieve further enrichment in phages carrying binding proteins. Following further bacterial-based amplification, the DNA in the interacting phage is sequenced to identify the interacting proteins or protein fragments. This sequence can be further mutagenized in vitro by site-specific mutagenesis (see Box 5.4) to optimize binding characteristics.

dna cloning in bacteria

Fundamental steps: •Vector •Insert •Insertion of the donor DNA into the vector -Restriction enzymes -Ligation -Transformation •Selection of the desired "clone" containing the insert

Why is Introduction of DNA into eukaryotic cells more difficult?

Introduction of DNA into eukaryotic cells is more complicated, especially for yeast and plant cells, which possess, in addition to the plasma membrane, outer protective poly- saccharide layers. These layers must be removed, usually by enzymatic treatments, to produce protoplasts, which survive and are competent for transformation.

liposome-mediated gene transfer

Liposomes are unilamellar (single-bilayer) vesicles prepared from cationic lipids that readily and spontaneously form complexes with DNA in solution. The positively charged liposomes also bind to cultured cells and presumably fuse with the plasma membranes. The use of liposomes for transformation or transfection is known as lipofection

all restriction enzymes require _____, and only __________ require ATP for functioning of the motor.

Mg2+ ; type 1

how to treat cells for transformation

Plasmid vectors are introduced into bacterial cells by transformation techniques that involve prior treatment of bacteria with calcium chloride at low temperature followed by a brief heat shock. It is believed that calcium chloride affects the structure of the cell wall and may help in the binding of DNA to the cell surface. The heat shock stimulates the actual DNA uptake.

whole-genome shotgun approach

Procedure for genome sequencing in which the genome is randomly cut into many overlapping short segments that are sequenced; computer software then assembles the complete sequence.

gene guns

Researchers have developed gene guns to facilitate gene transfer into cells that are notoriously difficult to transform. The method can be applied not only to unicellular organisms but also to plant leaves or entire animals, such as Drosophila and mice. It has been particularly useful for chloroplast transformation, as there are no other known methods to introduce foreign DNA into these organelles. The gene gun method relies on the ability of nucleic acids to adhere to biologically inert metal particles, such as gold or tungsten. The DNA-particle complex is accelerated under partial vacuum and hits the target tissue in its acceleration path. There are numerous ways to create acceleration, such as the use of pneumatic devices, magnetic or electrostatic forces, and sprayers.

typical binary vector and helper plasmid used for plant transformation.

The bacterial selectable marker and origin of replication are needed to produce the vector in the necessary purity and quantities in E. coli before transformation of the plant. Thevir genes that are needed for transfer and integration could be supplied in trans, on a second plasmid, thus forming a bi- nary transformation system with the vir helper plasmid generally contained a complete or partial deletion of the T-region, rendering strains containing this plasmid unable to incite tumors.

The initial binding of type II restrictases occurs

The initial binding of type II restrictases occurs at random sites, and the subsequent search for the recognition sequence involves one-dimensional translocation along the DNA.

vector capacity

Vector capacity is the size of the transgene(s) that could be packed into the vector. -It is determined by the size of the viral genome itself and by the amount of viral nucleic acid, DNA or RNA, that could be excised and replaced by a transgene without affecting viral trafficking ability.

viruses with what type of genome have higher transfection efficiency?

Viruses containing a dsDNA genome have, in general, higher transfection efficiency than ssDNA and RNA viruses bc the latter need to first convert their genomes into dsDNA in order to be incorporated to host

electroporation

When subjected to an electric shock—for example, brief exposure to a voltage gradient of 4000-8000 V/cm— cells take up exogenous DNA through holes created in the plasma membrane. Drugs such as colcemid that arrest cells at metaphase increase transformation efficiency.This increase is attributed to the lack of a nuclear membrane during this stage of the cell cycle, as the nuclear membrane also needs to be crossed for stable integration of the gene into the genome.

expression vectors

a DNA vector, such as a plasmid, that carries a DNA sequence for the expression of an inserted gene into mRNA and protein in a host cell as their name implies, these vectors are capable not only of carrying an inserted gene but also of expressing the protein product. They must ensure that the gene will be transcribed, and in a form that can be translated into protein, by use of the host cell's machinery.

hierarchical shotgun sequencing

also called BAC-based or clone-by-clone sequencing. a low-resolution physical map of the genome is made prior to actual sequencing. From this map, a minimal number of fragments that cover the entire chromosome are selected for sequencing.[15] In this way, the minimum amount of high-throughput sequencing and assembly is required. This approach involves generating and organizing, through restriction nuclease mapping, a set of large-insert clones, typically 100-200 kbp each, and then separately performing shotgun sequencing on the appropriately chosen clones.

reason why retroviruses can only affect rapidly dividing cells

crossing the nuclear membrane may be an insurmountable obstacle for some viruses, especially retroviruses. the elimination of the nuclear envelope during mitosis provides any exogenous DNA or RNA with a window of easy entry into the nucleus.

PCR steps

goal: amplify DNA fragment steps: denaturing annealing (to DNA primers) elongation (by heat stable DNA polymerase) agrose gel electrophoresis (separate by size)

Once in the cell, viral DNA ___________________.

is released from the capsid/envelope structures and needs to enter the nucleus. Crossing the nuclear membrane may be an insurmountable obstacle for some viruses, especially retroviruses. -The overall transfection efficiency depends on the efficiency of the steps involved in viral uptake, entry into the nucleus, and escape from degradation.

The best way to study which protein genes are transcribed, and later translated, in a given cell type is to

isolate the population of mature mRNA from the cytoplasm and then, by use of in vitro methods such as reverse transcription, to produce double- stranded DNA copies of this population. These are known as cDNAs or copy DNAs. Cloning this entire population of cDNAs into bacterial vectors produces cDNA librar- ies. These can be maintained stably in bacteria and stored indefinitely, exactly as genomic libraries are. The existence of such libraries has practical applications: because they lack introns, cDNA clones are a convenient source of sequences to be directly expressed in bacterial cells.

Trans- formed plant protoplasts are grown

on selective medium to produce large masses of undifferentiated cells known as calli, plural for callus. These can then be transferred to nutritional medium containing specific growth hormones that allow regeneration of whole fertile transformed plants.

Tropism

or host range, is the ability of the virus to infect different cell types.

The efficiency of introducing recombinant DNA molecules into bacterial cells is greater with

phage-based cloning vectors

One of the biggest problems in the development of gene therapy

the immunogenicity of the vector. On one hand, the immune response can very effectively eliminate transduced cells. On the other hand, the development of adaptive immunity against viral vectors can prevent their readministration.

Because the anionic nature of DNA hinders transfer across the membrane, the focus of efficient gene therapy is on

vector-mediated delivery

Type III restriction endonucleases

very similar to type I, but they do not require ATP, they methylate just one of the strands, and their cleavage site is relatively close to the recognition sequence.

2 types of vectors

viral and nonviral

procedure of Site-directed mutagenesis

• Synthesize a short, ~20-nt-long oligonucleotide sequence that complements the DNA sequence of the intended muta- tion site in the gene of interest but that carries deliberate mistakes, such as single-base substitutions, short deletions, or insertions at its center. • Anneal the oligonucleotide to a single-stranded cloned sequence of interest; the product will contain a base or sev- eral bases that cannot base-pair with the gene and, depend- ing on the change introduced in the oligonucleotide, may form a loop. The correctly matched bases on both sides of the mutation will keep the annealed complex stable. • DNA polymerase will use the annealed oligonucleotide as a primer to synthesize an entire strand complementary to the template, but retaining the modified oligonucleotide; DNA ligase will ligate the nick. • The new double-stranded circle is then introduced into bacterial cells and the bacteria are allowed to multiply. Various techniques can be used to get rid of the original molecule.