Chapter 4 Molecular Cloning Methods

1. Chapter 4Molecular Cloning Methods

2. Introduction The significance of gene cloning To elucidate the structure and function of genes. i.e.: investigating hGH gene hGH gene: <10-6 of human genome Problem 1: need kilograms of human genome DNA for 1μg hGH gene Problem 2: how to separate the gene from the rest DNA

3. 4.1 Gene Cloning The procedure in a gene cloning experiment is • To place a foreign gene into a bacterial cell; • To grow a clone of those modified bacteria. The principle factors for gene cloning experiment: • Restriction endonucleases • Vectors • Specific probe

4. The Role of Restriction Endonucleases • Vectors Plasmids as Vectors Phages as Vectors λ Phage Vectors Cosmids M13 phage vectors Phagemids Eukaryotic Vectors • Identifying a Specific Clone with a Specific Probe Polynucleotide Probes

5. 4.1.1 The Role of Restriction Endonucleases • Restriction : restrict the host range of the virus • Endonucleases : cut at sites within the foreign DNA • How to name: the first 3 letters of the Latin name of the microorganism + the strain designation + Roman numeral

8. The main advantage of restriction enzyme is there ability to cut a DNA reproducibly in the same place; this is the basis of many techniques used to analyze genes. Many restriction enzymes make staggered cut in the two DNA strands, leaving a sticky ends, that can base-pair together briefly.Enzymes that recognize identical sequences are called isoschizomers.

9. Restriction- modification systemR-M system Almost all restriction nucleases are paired with methylases that recognize and methylate the same DNA sites

10. Figure 4.1 Maintaining restriction endonuclease resistance after DNA replication We begin with an EcoRI site that is methylated (red) on both strands. After replication, the parental strand of each daughter DNA duplex remains methylated, but the newly made strand of each duplex has not been methylated yet. The one methylated strand in these hemimethylated DNAs is enough to protect both strands against cleavage by EcoRI. Soon, the methylase recognizes the unmethylated strand in each EcoRI site and methylates it, regenerating the fully methylated DNA.

12. Figure 4.2 The first cloning experiment involving a recombinant DNA assembled in vitro. Boyer and Cohen cut two plasmids, pSC101 and RSF1010, with the same restriction endonuclease, EcoRI. This gave the twolinear DNAs the same stickyends, which were then linked in vitro using DNA ligase. The investigators reintroduced the recombinant DNA into E. coli cells by transformation and selected clones that were resistant to both tetracycline and streptomycin. These clones were therefore harboring the recombinant plasmid.

13. Summary: Restriction endonucleases recognize specific sequences in DNA molecules and make cuts in both strands. This allows very specific cutting of DNAs. Also, because the cuts in the two strands are frequently staggered, restriction enzymes can create sticky ends that help link together two DNAs to form a recombinant DNA in vitro.

14. 4.1.2 Vectors Vectors serve as carriers to allow replication of recombinant DNAs. Origin of replication Multiple cloning site(MCS) Selection gene Plasmids pBR322 pUC Phages λphage cosmids M13 Phagemids

15. Plasmids as Vectors

16. Summary: The first generations of plasmid cloning vectors were pBR322 and the pUC plasmids. The former has two antibiotic resistance genes and a variety of unique restriction sites into which one can introduce foreign DNA. Most of these sites interrupt one of the antibiotic resistance genes, making screening straightforward. Screening is even easier with the pUC plasmids. These have an ampicillin resistance gene and a multiple cloning site that interrupts a partial β-galactosidase gene. One screens for ampicillin-resistant clones that do not make active β-galactosidase and therefore do not turn the indicator, X-gal, blue. The multiple cloning site also makes it convenient to carry out directional cloning into two different restriction sites.

17. Figure 4.3 The plasmid pBR322, showing the locations of 11 unique restriction sites that can be used to insert foreign DNA The locations of the two antibiotic resistance genes (Ampr =ampicillin resistance; Tetr =tetracycline resistance) and the origin of replication (ori ) are also shown. Numbers refer to kilobase pairs (kb) from the EcoRI site.

19. Figure 4.4 Cloning foreign DNA using the PstI site of pBR322. We cut both the plasmid and the insert (yellow) with PstI, then join them through these sticky ends with DNA ligase. Next, we transform bacteria with the recombinant DNA and screen for tetracycline-resistant, ampicillin-sensitive cells. The recombinant plasmid no longer confers ampicillin resistance because the foreign DNA interrupts that resistance gene (blue).

20. Figure 4.5 Screening bacteria by replica plating. (a) The replica plating process. We touch a velvet-covered circular tool to the surface of the first dish containing colonies of bacteria. Cells from each of these colonies stick to the velvet and can be transferred to the replica plate in the same positions relative to each other. (b) Screening for inserts in the pBR322 ampicillin resistance gene by replica plating. The original plate contains tetracycline, so all colonies containing pBR322 will grow. The replica plate contains ampicillin, so colonies bearing pBR322 with inserts in the ampicillin resistance gene will not grow (these colonies are depicted by dotted circles). The corresponding colonies from the original plate can then be picked.

22. pUC

23. lacZ’ : coding for the amino terminalportion of the enzyme β –galactosidease. Host E.coli strain carry a gene fragment that codes the carboxyl potion of β –galactosidease; When X-gal cleaved by β –galactosidease, it releases galactose plus an indigo dye that stains the bacterial colony blue.

24. Figure 4.7 Joining of vector to insert. (a) Mechanism of DNA ligase. Step 1: DNA ligase reacts with an AMP donor—either ATP or NAD(nicotinamide adenine dinucleotide), depending on the type of ligase. This produces an activated enzyme (ligase-AMP). Step 2: The activated enzyme donates a phosphate to the free 5’-phosphate at the nick in the lower strand of the DNA duplex, creating a high-energy diphosphate group on one side of the nick. Step 3: With energy provided by cleavage of the diphosphate, a new phosphodiester bond is created, sealing the nick in the DNA. This reaction can also occur in both DNA strands at once, so two independent DNAs can be joined together by DNA ligase.

25. Figure 4.7 Joining of vector to insert. (b)Alkaline phosphatase prevents vector re-ligation. Step 1: We cut the vector(blue, top left) with BamHI. This produces sticky ends with 5’-phosphates(red). Step 2: We remove the phosphates with alkaline phosphatase, making it impossible for the vector to re-ligate with itself. Step 3: We also cut the insert(yellow, upper right) with BamHI, producing sticky ends with phosphates that we do not remove. Step4: Finally, we ligate the vector and insert together. The phosphates on the insert allow two phosphodiester bonds to form(red), but leave two unformed bonds, or nicks, These will be completed once the DNA is in the transformed bacterial cell.

26. Phages as vectors Natural advantages over plasmid: They infect cells much more efficiently than plasmids transform cells, so the yield of clones with phage vectors is usually higher.

27. Summary: Two kinds of phages have been especially popular as cloning vectors. The first of these is λ, from which certain nonessential genes have been removed to make room for inserts. Some of these engineered phages can accommodate inserts up to 20 kb, which makes them useful for building genomic libraries, in which it is important to have large pieces of genomic DNA in each clone. Cosmids can accept even larger inserts—up to 50 kb—making them a favorite choice for genomic libraries. The second major class of phage vector is composed of the M13 phages. These vector have the convenience of a multiple cloning site and the further advantage of producing single-stranded recombinant DNA, which can be used for DNA sequencing and for site-direct mutagenesis. Plasmids called phagemids have also been engineered to produce single-stranded DNA in the presence of helper phages.

28. Figure 4.8 Cloning in Charon 4. (a)Forming the recombinant DNA. We cut the vector (yellow) with EcoRI to remove the stuffer fragment and save the arms. Next, we ligate partially digested insert DNA (red) to the arms. (b) Packaging and cloning the recombinant DNA. We mix the recombinant DNA from (a) with an in vitro packaging extract that contains λ phage head and tail components and all other factors needed to package the recombinant DNA into functional phage particles. Finally, we plate these particles on E.coli and collect the plaques that form.

29. Figure 4.9 Selection of positive genomic clones by plaque hybridization. First, we touch a nitrocellulose ot similar filter to the surface of the dish containing the Charon 4 plaques from Figure 4.8. Phage DNA released naturally from each plaque will stick to the filter. Next, we denature the DNA with alkali and hybridize the filter to a labeled probe for the gene we are studying, then use X-ray film to reveal the position of the label. Cloned DNA from one plaque near the center of the filter has hybridized, as shown by the dark spot on the film.

30. Cosmids Behave both as plasmids and as phages; Contain the cos sites of λ and plasmid origin of replication; Have room for 40-50 kb inserts.

31. M13 phage vectors β –galactosidease gene fragment pUC family MCS Single stranded DNA genome

32. Figure 4.10 Obtaining single-stranded DNA by cloning in M13 phage. Foreign DNA (red), cut with HindIII, is inserted into the HindIII site of the double-stranded phage DNA. The resulting recombinant DNA is used to transform E.coli cells, whereupon the DNA replicates by a rolling circle mechanism, producing many single-stranded product DNAs. The product DNAs are called positive (+) strands, by convention. The template DNA is therefore the negative (-) strand.

33. Phagemides Single-stranded; Both phage and plasmid characteristics; Help phage Two RNA polymerase promoters (T7and T3)

35. Summary Two kinds of phages have been especially popular as cloning vectors. The first of these is λ, from which certain nonessential genes have been removed to make room for inserts. Some of these engineered phages can accommodate inserts up to 20 kb, which makes them useful for building genomic libraries, in which it is important to have large pieces of genomic DNA in each clone. Cosmids can accept even larger inserts—up to 50 kb—making them a favorite choice for genomic libraries. The second major class of phage vector is composed of the M13 phages. These vector have the convenience of a multiple cloning site and the further advantage of producing single-stranded recombinant DNA, which can be used for DNA sequencing and for site-direct mutagenesis. Plasmids called phagemids have also been engineered to produce single-stranded DNA in the presence of helper phages.

36. 4.1.3 Identifying a Specific Clone with a Specific Probe Polynucleotide Probes High stringency Low stringency

37. Summary Specific clones can be identified using polynucleotide probes that bind to the gene itself. Knowing the amino acid sequence of a gene product, one can design a set of oligonucleotides that encode part of this amino acid sequence. This can be one of the quickest and most accurate means of identifying a particular clone.

38. 4.2 The Polymerase Chain Reaction (PCR) PCR amplifies a region of DNA between two predetermined sites. Oligo-nucleotides complementary to these sites serve as primers for synthesis of copies of the DNA between the sites. Each cycle of PCR double the number of copies of the amplified DNA until a large quantity has been made.

41. Figure 4.12 Amplifying DNA by the polymerase chain reaction. Cycle 1: Start with a DNA duplex (top) and heat it to separate its two strands (red and blue). Then add short, single-stranded DNA primers (purple and yellow) complementary to sequences on either side of the region (X) to be amplified. The primers hybridize to the appropriate sites on the separated DNA strands; now a special heat-stable DNA polymerase uses these primers to start synthesis of complementary DNA strands. The arrows represent newly made DNA, in which replication has stopped at the tip of the arrowhead. At the end of cycle 1, two DNA duplexes are present, including the region to be amplified, whereas we started with only one. The 5’→3’ polarities of all DNA strands and primers are indicated throughout cycle 1. The same principles apply in cycle 2. Cycle 2: Repeat the process, heating to separate DNA strands, cooling to allow annealing with primers, and letting the heat-stable DNA polymerase make more DNA. Now each of the four DNA strands, including the two newly made ones, can serve as templates for complementary DNA synthesis. The result is four DNA duplexes that have the region to be amplified. Notice that each cycle doubles the number of molecules of DNA because the products of each cycle join the parental molecules in serving as templates for next cycle. This exponential increase yields 8 molecules in the next cycle and 16 in the cycle after that. This process obviously leads to very high numbers in only a short time.

42. 4.2.1 cDNA Cloning • Nick translation • Reverse transcriptase • RNase H • Terminal transferase

43. Figure 4.13 Making a cDNA library. This figure focuses on cloning a single cDNA , but the method can be applied to a mixture of mRNAs and produce a library of corresponding cDNAs. (a) Use oligo(dT) as a primer and reverse transcriptase tocopy the mRNA (blue), producing a cDNA (red) that is hybridized to the mRNA template. (b) Use RNase H to partially digest the mRNA, yielding a set of RNA primers base-paired to the first-strand cDNA. (c) Use E.coli DNA polymerase I under nick translation conditions to build second-strand cDNAs on the RNA primers. (d) The second-strand cDNA growing from the leftmost primer (blue) has been extended all the way to the 3’-end of the oligo(dA) corresponding to the oligo(dT) primer on the first-strand cDNA. (e) To give the double-stranded cDNA sticky ends, add oligo(dC) with terminal transferase. (f) Anneal the oligo(dC) ends of the cDNA to complementary oligo(dG) ends of a suitable vector (black). The recombinant DNA can then be used to transform bacterial cells. Enzymes in these cells remove remaining nicks and replace any remaining RNA with DNA.

45. Figure 4.15 Using RT-PCR to clone a single cDNA. (a) Use a reverse primer (red) with a HindIII site (yellow) at its 5’-end to start first-strand cDNA synthesis, with reverse transcriptase to catalyze the reaction. (b)Denature the mRNA-cDNA hybrid and anneal a forward primer (red) with a BamHI site (green) at its 5’-end. (c) This forward primer initiates second-strand cDNA synthesis, with DNA polymerase catalyzing the reaction. (d)Continue PCR with the same two primers to amplify the double-stranded cDNA. (e) Cut the cDNA with BamHI and HindIII to generate sticky ends. (f)Ligate the cDNA to the BamHI and HindIII sites of a suitable vector (purple). Finally, transform cells with the recombinant cDNA to produce a clone.

46. Figure 4.16 RACE procedure to fill in the 5’-end of a cDNA. (a) Hybridize an incomplete cDNA (red), or an oligonucleotide segment of a cDNA to mRNA (green), and use reverse transcriptase to extend the cDNA to the 5’-end of the mRNA. (b) Use terminal transferase and dCTP to add C residues to the 3’end of the extended cDNA; also, use RNase H to degrade the mRNA. (c) Use an oligo(dG) primer and DNA polymerase to synthesize a second strand of cDNA (blue). (d) Perform PCR with oligo(dG) as the forward primer and an oligonucleotide that hybridizes to the 3’-end of the cDNA as the reverse primer. (e)The product is a cDNA that has been extended to the 5’-end of the mRNA. A similar procedure (3’-RACE) can be used to extend the cDNA in the 3’-direction. In that case, there is no need to tail the 3’-end of the cDNA with terminal transferase because the mRNA already contains poly(A); thus, the reverse primer would be oligo(dT).

47. Summary To make a cDNA library, we can synthesize cDNAs one strand at a time, using mRNAs from a cell as templates for the first strands and these first strands as temletes for the second strands. Reverse trnscriptase generates the first strands and E.coli DNA polymerase I generates the second strands. We can endow the double stranded cDNAs with oligonucleotide tails that base-par with complementary tails on a cloning vector. We can then use these recombinant DNAs to transform bacteria. We can use RT-PCR to generate a cDNA from a single type of mRNA, but we must know the sequence of the mRNA in order to design the primers for the PCR step. If we put restriction sites on the PCR primers, we place these sites at the ends of the cDNA,so it is easy to ligate the cDNA into a vector. We can detect particular clones by colony hybridazation with redioactive DNA probes,or with antibodies if an expression vector such as λgt11 is used.

48. 4.3 Methods of Expressing Cloned Genes

49. 4.3.1 Expression Vectors • Expression vectors with strong promoters • Inducible Expression Vectors • Expression vectors produce fusion proteins • Eukaryotic expression vectors

50. Figure 4.17 Producing a fusion protein by cloning in a pUC plasmid. Insert foreign DNA (yellow) into the multiple cloning site (MCS); transcription from the lac promoter (purple) gives a hybrid mRNA beginning with a few lacZ’ codons, changing to insert sequence, then back to lacZ’ (red). This mRNA is translated to a fusion protein containing a few β-galactosidase amino acids for the remainder ofthe protein. Because the insert contains a translation stop codon, the remaining lacZ’ codons are not translated. Figure 4.17