Gene library.ppt is a good start to learn
- 1. Construction of Gene Library Tileye Feyissa
- 2. The isolation of genes that encode proteins is often the goal of a biotechnology experiment In prokaryotes, structural genes have continuous coding domain In eukaryotes, the exons are separated by introns Therefore, different cloning strategies have to be used
- 5. To achieve this, the complete DNA of an organism is cut with a restriction enzyme Each fragment is inserted into a vector Then the specific cell line (clone) that carries the target DNA sequence must be identified, isolated, and characterized This process of subdividing genomic DNA into clonable elements and inserting them into host cells is called creating a library (clone bank, gene bank) In a prokaryote, the target DNA is frequently minuscule, 0.02% The problem is then how to clone and select the targeted DNA sequence
- 6. One way to create DNA library is by treating the DNA from a source organism with a four-cutter restriction endonuclease (e.g. Sau3AI) This enzyme theoretically cleaves the DNA approximately once in every 256 bp The conditions of the digestion reactions are set to give a partial, not a complete digestion In this way, all possible fragment sizes are generated
- 7. However, restriction endonuclease sites are not randomly located Some fragments may be too large to be cloned In these cases, an incomplete library is available for selection So it may be difficult or even impossible to find a specific target DNA sequence This problem can be overcome by forming a library with another restriction enzyme After a library is created, the clones with the target sequence must be identified
- 8. Screening DNA library Three popular methods of identification are used - DNA hybridization with a labeled DNA probe - Immunological screening for the protein product - Screening for protein activity
- 9. Screening by DNA hybridization The presence of a target DNA sequence can be determined by this method It depends on the formation of stable base pairs between the probe and the target sequence Double stranded DNA can be converted into single-stranded DNA by heat or alkali treatment The target DNA is denatured and the single strands are irreversibly bound to a matrix (nitrocellulose or nylon) This binding is carried out at a high temperature
- 10. Then the single strands of a DNA probe are incubated with the bound DNA sample The probes are labeled with either a radioisotope or another tagging system If the sequences of nucleotide in the DNA probe is complementary to a nucleotide sequence in the sample, then hybridization occurs
- 11. The hybridization can be detected by autoradiography or other visualization procedures, depending on the nature of the probe label If the nucleotide sequence of the base does not base pair with a DNA sequence in the sample Then no hybridization occurs and the assay gives a negative result
- 12. Probes range in length from 100 to more than 1000 bp Both larger and smaller probes can be used Depending on the conditions of the hybridization rxn, stable base pairing requires a match of > 80% within a segment of 50 bases
- 13. DNA probes can be labeled in various ways - One strategy is by random primer method - This method utilizes a mixture of synthetic random oligonucleotides - These oligonucleotides are formed from all possible combinations sequences of six nucleotides that act as primers for DNA synthesis - On the bases of the chance occurrence of complementary sequences, some of the oligomers in the sample will hybridize to complementary sequences on the unlabeled probe DNA template
- 14. After the oligomer sample is mixed with the denatured probe template DNA, The four dNTPs and the Klenow fragment are added The Klenow fragment retains both DNA polymerase and 3’ exonuclease activities but lacks the 5’ exonuclease activity that is normally associated with E. coli DNA polymerase I With the available 3’-OH groups of the bound random primers and the strands of the probe as templates, new DNA synthesis occurs If a radioactive label is used, then one of the dNTPs contains the isotope 32P in the -position phosphate Autoradiography can be used to determine whether the labeled probe sequences hybridize to sequences of a target DNA sample
- 16. For nonisotopic detection of hybridization biotin digoxygenin horse radish peroxidase can be attached to one of the four dNTPs that is incorporated during the DNA synthesis step
- 18. When a probe with this kind of label hybridizes to the sample DNA, Detection is based on the binding of an intermediary compound (e.g. streptavidin) that carries an appropriate enzyme Depending on the assay system, the enzyme can be used for the formation of either a chromogenic molecule that can be visualized directly or A chemiluminescent response that can be detected by autoradiography
- 19. There are at least two possible sources of probes for screening a genomic library 1. Cloned DNA from a closely related organism (a heterologous probe) can be used - In this case, the conditions of the hybridization rxn can be adjusted to permit considerable mismatch between the probe and the target DNA to compensate for the natural differences between the two sequences 2. A probe can be produced by chemical synthesis - The nucleotide sequence of a synthetic probe is based on the probable nucleotide sequence that is deduced from the known amino acid sequence of the protein encoded by the target gene
- 20. Genomic DNA libraries are often screened by plating out the transformed cells on the growth medium of a master plate Then transferring samples of each colony to a solid matrix such as a nitrocellulose or nylon membrane The cells are lysed, deproteinized, the DNA is denatured and bound to the matrix At this stage, a labeled probe is added If hybridization occurs, signals are observed on an autoradiograph The colonies from the master plate that correspond to samples containing hybridized DNA are then isolated and cultured
- 24. Because most libraries are created from partial digestions, a number of colonies (clones) may give a positive response to the probe The next task is to determine which clone, if any, contains the complete sequence of the target gene Preliminary analyses that use the results of gel electrophoresis and restriction endonuclease mapping reveal the length of each insert and identify those inserts that are the same and those that share overlapping sequences By using overlapping sequences, it may be possible to join sections of the gene in additional cloning exp’ts
- 25. Alternatively, if an insert in any one of the clones is large enough to include the full gene, Then the complete gene can be recognized after DNA sequencing Because this will have start and stop codons and contiguous set of nucleotides that code for the target protein Unfortunately, there is no guarantee that the complete sequence of a target gene will be present in a particular library If the search for an intact gene fails, then another library can be created with a different restriction endonuclease and screened with either the original probe or probes derived from the first library
- 26. Alternatively, libraries that contain DNA fragments larger than the average prokaryotic gene can be created to increase the chance that some members of the library will carry a complete version of the target gene
- 27. Screening by immunological assay If a DNA probe is not available, this is alternative method to screen a library E.g. if a cloned DNA sequence is transcribed and translated, The presence of the protein, or even parts of it, can be determined by an immunological assay All cell lines of the library are grown on master plates A sample of each colony is transferred to a matrix, where the cells are lysed and the released proteins attach to the matrix The matrix with the bound proteins is treated with an antibody (primary antibody)
- 28. The primary antibody specifically binds to the protein encoded by the target gene Following the interaction of the primary antibody with the target protein (antigen), Any unbound antibody is washed away and the matrix is treated with a second antibody that is specific for the primary antibody The secondary antibody has an enzyme such as alkaline phosphatase attached to it After the matrix is washed, a colorless substance is added
- 29. If the secondary antibody has bound to the primary antibody, The colorless substrate is hydrolyzed by the attached enzyme and produces a colored compound that accumulates at the site of the rxn The colonies on the master plate that correspond to positive results (colored spots) on the matrix contain either an intact gene or a portion of the gene that is large enough to produce a protein product that is recognized by the primary antibody After detection by immunoassay of genomic DNA libraries, The positive clones must be characterized further to determine which, if any, carry a complete gene
- 30. Screening by protein activity If the target gene produces an enzyme that is not normally made by the host cell, A plate assay can be devised to identify members of a library that carry the functional gene encoding that enzyme E.g. the genes for -amylase, endoglucanase, and -glucosidase from various organisms have been isolated by plating the genomic library in E. coli onto medium supplemented with a specific substrate
- 31. Then using a selective stain to identify those colonies that are capable of utilizing the substrate The gene that is sought encodes the product that is essential for the growth of a mutated host cell, then the library can be formed by transformation into these mutant cells
- 32. The cells that are able to grow on minimal medium in the absence of the required substrate must carry a functional form of the target gene on the cloning vector Variations of this form of genetic complementation have been used to isolate a variety of important genes These genes include those for biosynthesis of antibiotics and the formation of nitrogen-fixing nodules on the roots of certain plants
- 33. Cloning DNA sequences that encode eukaryotic proteins Special techniques are required for cloning eukaryotic structural genes Prokaryotic hosts are not able to remove introns from transcribed RNA Therefore, this mRNA is not translated correctly in a bacterial host cell A eukaryotic DNA sequence needs prokaryotic transcriptional and translational control sequences to be properly expressed
- 34. A functional eukaryotic mRNA has a G cap at the 5’ end and usually a string of up to 200 adenine residues (poly (A) tail) The poly (A) tail can be used to separate the mRNA fraction of a tissue from the rRNA and tRNA Extracted cellular eukaryotic RNA is passed through a column packed with cellulose beads to which is bound short chains of thymidine residues Each thymidine residues are about 15 nucleotides long (Oligo(dT), dT15) The poly(A) tails of the mRNA bind by base pairing to the oligo(dT) chains The tRNA and rRNA which lack poly(A) tails, pass through the column
- 35. The mRNA is eluted from the column by treatment with a buffer that breaks the A:T hydrogen bonds Thereby releasing the bound mRNA Before the mRNA can be cloned into a vector, They must be converted to double-stranded DNA
- 36. This synthesis is accomplished by using two different kinds of nucleic acid polymerases Reverse transcriptase and Klenow fragment of DNA pol I After the mRNA fraction is purified, short unbound sequences of oligo(dT) molecules enzyme reverse transcriptase and the four dNTPs are added to the sample The oligo(dT) base pair with the poly(A) tail regions and provide an available 3’-hydroxyl group to prime the synthesis of a DNA strand Reverse transcriptase uses RNA strand as a template
- 37. The synthesis of DNA strand by reverse transcriptase in vitro is often incomplete However, before synthesis ceases, the DNA strand usually turns back on itself for a few nucleotides to form a hairpin loop The second DNA strand is synthesized by the addition of the Klenow fragment of E.coli DNA pol which uses the first DNA strand as a template After the rxn is complete, the sample is treated with the enzyme RNaseH RNaseH degrades the mRNA S1 nuclease opens the hairpin loops and degrades single-stranded DNA extensions
- 38. At the end of this procedure, the sample contains a mixture of partial and complete double-stranded complementary (cDNA) copies of the more prevalent mRNAs in the original sample This cDNA populations can be cloned by blunt-end ligation or other joining mechanisms into a plasmid cloning vector to form a cDNA library Then screen the DNA library by one of the screening methods
- 39. DNA manipulative enzymes The range of DNA manipulative enzymes can be grouped into five broad classes This classification is based on the type of rxn that they catalyze 1. Nucleases 2. Ligases 3. Polymerases 4. Modifying enzymes 5. Topoisomerases
- 40. Although most enzymes can be assigned to a particular class, a few display multiple activities that span two or more classes Many polymerases combine their ability to make new DNA molecules with an associated nuclease activity Many similar enzymes able to act on RNA are known (e.g. ribonuclease)
- 41. Gene cloning requires that DNA be cut in a very precise and reproducible fashion Each vector must be cleaved at a single position to open up the circle so that new DNA can be inserted It is also necessary to cleave the DNA that is to be cloned There are two reasons for this Restriction endonucleases
- 42. 1. If the aim is to clone a single gene, which may consist of only 2 or 3 kb of DNA, that gene will have to be cut out of the large DNA 2. Large DNA may have to be broken down to produce fragments of small enough to be carried by the vector
- 43. Blunt ends and sticky ends The exact nature of the cut produced by a restriction endonuclease is of considerable importance in design of a gene cloning expt Many restriction endonucleases make a simple double-stranded cut in the middle of the recognition sequence, resulting in blunt or flush end e.g. PVuII and AluI
- 44. A large number of restriction endonucleases cut DNA in a slightly different way The cleavage is staggered by two or four nucleotides resulting in short single-stranded overhungs at each end These are called sticky or cohesive ends The restriction endonucleases with different recognition sequences may produce the same sticky ends. E.g. BamHI, BgllI, Sau3A
- 47. Restriction Sites are not evenly spaced
- 48. Sticky ends increase the efficiency of ligation Although ligation of two blunt-ended fragments can be carried out in the test tube, it is not very efficient This is because ligase is unable to ‘catch hold’ of the molecule to be ligated Has to wait for chance association to bring the ends together If possible, blunt end ligation should be performed at high DNA concentrations In contrast, ligation of complementary sticky ends is much more efficient
- 49. Compatible sticky ends can base pair with one another by hydrogen bonding Forming a relatively stable structure for the enzyme to work on If phosphodiester bonds are not synthesized fairly quickly, the sticky ends will fall apart again These transient, base-paired structures do, however, increase the efficiency of ligation by increasing the length of time the ends are in contact with one another
- 51. Putting sticky ends onto blunt-ended molecule Compatible sticky ends are desirable on the DNA molecules to be ligated together in a gene cloning exp’t These sticky ends can be provided by digesting both the vector and the DNA to be cloned with the same restriction endonucleases, or With different enzymes that produce the same sticky end However, it is not always possible to do this A common situation is where the vector molecule has sticky ends, but the DNA fragments to be cloned are blunt-ended Under these circumstances, one of three methods can be used to put the correct sticky ends onto the DNA fragments
- 52. Linkers Are short pieces of double-stranded DNA Of known nucleotide sequence Are synthesized in the test tube It is blunt-ended, but contains a restriction site (e.g. BamHI) DNA ligase can attach linkers to the ends of larger blunt-ended DNA
- 53. This particular rxn can be performed very efficiently Synthetic oligonucleotides such as linkers can be made in a very large amounts and added into the ligation mix at a high concentration More than one linker will attach to each end of the DNA molecule, producing the chain structure
- 54. However, digestion with BamHI cleaves the chains at the recognition sequences Producing a large number of cleaved linkers and the original DNA fragment, now carrying BamHI sticky ends This modified fragment is ready for ligation into a cloning vector restricted with BamHI There is one potential drawback with the use of linkers
- 56. Consider what would happen if the blunt-ended molecule contained one or more BamHI recognition sequences If this was the case, the restriction step needed to cleave the linkers and produce sticky ends would also cleave the blunt-ended molecule The resulting fragments will have correct sticky ends, But that is no consolation if the gene contained in the blunt-ended fragment has now been broken into pieces
- 58. The second method of attaching sticky ends to blunt-ended molecule is designed to avoid this problem Adaptors Short synthetic oligonucleotides Has one sticky end The idea is to ligate the blunt end of the adaptor to the blunt ends of the DNA fragment, to produce a new molecule with sticky ends This may appear to be simple method but in practice a new problem arises The sticky ends of individual adaptor molecules could base pair with each other to form dimers So that the new DNA molecule is still blunt-ended
- 61. The sticky ends could be recreated by digestion with a restriction digestion But that would defeat the purpose of using adaptors in the first place The answer to the problem lies in the precise chemical structure of the ends of the adaptor molecule One end, 5’ terminus, carries a phosphate group (5’-P) The other, the 3’ terminus, has a hydroxyl group (3’-OH) In the double helix the two strands are antiparallel So each end of a double-stranded molecule consists of one 5’-P terminus and one 3’-OH terminus Ligation normally takes place between the 5’-P and 3’-OH ends
- 63. Adaptor molecules are synthesized so that the blunt end is the same as the ‘natural’ DNA, but the sticky end is different The 3’-OH terminus of the sticky end is the same as usual, but the 5’-P terminus is modified It lacks the phosphate group, and a 5’-OH terminus
- 64. DNA ligase is unable to form phosphodiester bridge between 5’-OH and 3’-OH ends The result is that, although base pairing is always occurring between the sticky ends of adaptor molecules, the association is never stabilized by ligation Adaptors can therefore be ligated to a DNA molecule but not to themselves After the adaptors have been attached, the abnormal 5’-OH terminus is converted to the natural 5’-P form by treatment with the enzyme polynucleotide kinase, Producing a sticky-ended fragment that can be inserted into an appropriate vector
- 66. Producing sticky ends by homopolymer tailing The technique of homopolymer tailing offers a radically different approach It is a polymer in which all the subunits are the same A DNA strand made up entirely of, say, deoxyguanosine is an example of a homopolymer, and is referred to as polydeoxyguanosine or poly (dG) Tailing involves using the enzyme terminal deoxynucleotidyl transferase to add a series of nucleotides onto the 3’-OH termini of a double-stranded DNA If this rxn is carried out in the presence of just one deoxyribonucleotide, a homopolymer tail is produced To be able to ligate two tailed molecules together, the homopolymers must be complementary
- 68. Frequently poly(dC) tails are attached to the vector and poly(dG) to the DNA to be cloned Base pairing between the two occurs when the DNA molecules are mixed In practice, the poly(dG) and poly(dC) tails are not usually exactly the same length, and the base-paired recombinant molecules that result have nicks as well as discontinuities
- 69. Repair is therefore, a two step process using Klenow polymerase to fill in the nicks followed by DNA ligase to synthesize the final phosphodiester bonds This repair rxn does not always have to be performed in the test tube If the complementary homopolymer tails are longer than about 20 nucleotides, then quite stable base-paired associations are formed
- 71. A recombinant DNA molecule, held together by base pairing although not completely ligated, is often stable enough to be introduced into the host cell in the next stage of the cloning exp’t Once inside the host, the cell’s own DNA polymerase and DNA ligase repair the recombinant DNA molecule, completing the construction begun in the test tube