Exercise no. 8 DNA Vectors - Worksheet 4 - Molecular Biology - PDF

WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Exercise no. 4 DNA vectors (plasmids) as the basic tool of molecular biology and genetic engineering – elements of molecular cloning, aspects of DNA restriction analysis. Theory Student:...

WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Exercise no. 4 DNA vectors (plasmids) as the basic tool of molecular biology and genetic engineering – elements of molecular cloning, aspects of DNA restriction analysis. Theory Student:  knows the basic terminology regarding molecular cloning  knows the basic terminology regarding DNA restriction analysis. Plasmid - DNA vector Plasmids are, additional non-chromosome DNA elements that control their own replication. They are mostly found in bacteria and some eukaryotes such as yeast or algae. Plasmids are generally circular particles of a double DNA strand with dimensions between 1kb to over 100 kb. In comparison to bacterial chromosomes plasmids are relatively very small. The number of plasmid copies in a cell is strictly specified and depends on the type of ORI (Origin of Replication) sequence, meaning the so-called sequence of the beginning of particle replication. Smaller plasmids often have a greater number of copies. Some classes of plasmids have the ability of horizontal transfer between bacteria of various species. The genetic material of plasmids in necessary for the life-cycle of bacteria, however it often contains antibiotic resistance genes, providing the bacteria with an advantage in a hostile environment. Restriction enzymes – DNA „molecular scissors” Restriction enzymes have the properties of nucleases meaning enzymes which “cut the DNA” hydrolyzing the phosphodiester bond between subsequent bases in the DNA strand. They are present in bacteria and cyanobacteria (prokaryotic organisms), where in combination with specific methylases they make up the so-called restriction-modification system. This system protects the cell from penetration from foreign DNA (e.g. from DNA bacteriophages). It consists of a set of two types of enzymes that recognize and modify the same DNA sequence:  restriction endonuclease (cleaves DNA),  methyltransferase (adds methyl groups to DNA). Modification through methylation protects the DNA from an attack by its own restriction enzymes. In turn, foreign DNA (e.g. phage, plasmid) has a recognizable non-methyl sequence – and then it is degraded. Bacteria used for cloning have a modified restriction - modification system (the so-called hdsR mutants – which have no restriction ability, but maintain their methylation activity). Page 1 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Types of restriction enzymes Type I – compound, contain many sub-particles, cleave the DNA at random locations far from the recognition sequence (up to 1000 nucleotides). They often contain a combination of restriction-modification enzymes. Type II – cleave the DNA at specific location, near or in the recognition palindromic sequence (that is why, they are most frequently used in molecular cloning). The IIs subtypes are enzymes, that cleave the DNA at specific (several/several tens nucleotides) distances from the recognition sequence, always on one side. Type III is similar to type I, however these enzymes, in order to recognize the sequence, require to non-palindromic sequences with opposite orientation placed near each other on the same DNA molecule. They cleave the DNA at random locations far from the recognition sequence (up to several tens of nucleotides). Restriction enzyme nomenclature The names of enzymes are written in cursive and are generally created from the first letter of the generic name of the host and the first two letters of the specific name. Following these letters there may be several other letters or Arabic numerals specifying the bacteria strain. The name ends with a Roman numeral, specifying the order in which a given enzyme was isolated from a given strain Sequences recognized by restriction enzymes Restriction enzymes, especially type II enzymes, commonly recognize palindromic sequences, of various lengths, between four to eight and more bases. In the human genome and in the higher eukaryotes:  enzymes that recognize four nucleotide sequences generate fragments with an average length of 260 bp,  enzymes that recognize six nucleotide sequences digest DNA into fragments with an average length of 4100 bp  enzymes that recognize six nucleotide sequences, that are specific for sequences containing GC, which yields fragments with an average length of 65000 bp. The lengths of fragments depend on the presence of th appropriate recognition sequences. The longer the sequence, the more seldom is it present in the genome, but they are not dispersed randomly. That is why various sequences digested by the same enzyme yield fragments of various lengths. Due to the specificity of the cleaving a given DNA sequence is always cleaved in the same locations by a specific type II restriction enzyme. In such a case we may talk about repetition of enzymatic cleaving. Page 2 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Type II restriction enzymes are made up of two identical subunits (homodimers), and each of them has one active center recognizing and cleaving one of the DNA strands. Depending on the placement of the active centers in relation to each other and the substrate (DNA) the cleavage points may be directly opposite each other – then the DNA ends have all bases paired (blunt ends) or the cleavage points may be moved a few base pairs in relation to each other (in palindromic sequences always symmetrically) – then we obtain DNA having sticky ends:  3’(if the unpaired fragments have a 3’–OH group at the end),  5’ (if the unpaired fragments have a 5’–P group at the end) (Fig.1). The 3’–OH i 5’–P endings are joined together by DNA ligase. Fig. 1.Examples of type II restriction enzymes yielding blunt and sticky ends 5’ Specificity of restriction enzymes may be examined because of:  Specific recognition of a DNA sequence,  Specific cleaving of a DNA sequence. Specific recognition of a given sequence is a property of all types of restriction enzymes, while the specificity of DNA cleaving pertains only to type II restriction enzymes. Only restrictive endonucleases, that cleave DNA specifically in relation to the sequence, ensure repetition of DNA cleaving into fragments – that is why they are broadly used in genetic engineering. Restriction enzymes change their structure when they are near a recognition sequence (Fig 2). Page 3 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Structure of BamHI dimer near a non-specific sequence Structure of a BamHI dimer at the site of a specifically recognized sequence 5'---G-3’ 5’-GATCC---3' Method of cleaving ( ) by the BamHI dimer (sticky ends 5’) 3'---CCTAG-5’ 3’-G---5' Fig.2. The BamHI enzyme cleaves a recognized sequence (5’GGATCC / 3’ CCTAGG). A recognized sequence allows for a closer binding of the enzyme with the DNA – thus enabling joining the active center with the cleaved phosphodiester bond. Artificial restriction enzymes With the use of genetic engineering methods fusion proteins were created consisting of a natural or synthetic domain specifically binding DNA sequences as well as a nuclease domain type II or IIs. Such enzymes specifically recognized longer DNA fragments (up to 36 bp) and are ultimately to be used for clinical (removal of damaged genes, introducing genes into the appropriate location in the genome) and industrial purposes. The information regarding all known restrictases may be found online in the Restriction Enzyme Database at http://rebase.neb.com. Other enzymes used in molecular cloning Additional enzymes used in cloning are enzymes with exonuclease or polymerase activity respectively cutting off single DNA strands or rebuilding a missing strand. S1 nuclease digests DNA, as well as RNA, but only in a single-strand form – after the digestion it leaves the 3’ –OH and 5’ –P ends. Page 4 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 The Klenow Fragment is a fragment of DNA polymerase with polymerase activity and a maintained exonuclease activity, which cuts off the protruding 3’ sticky ends or adds the missing 3’ strand to the protruding 5’ sticky end (direction of synthesis from 5’ to 3’), thus making the sticky DNA ends into blunt ones. T4 ligase – the enzyme comes from the T4 bacteriophage and it catalyzes the creation of a covalent phosphodiester bond between the 5’-P phosphate group of one strand and the 3-OH hydroxyl group of another strand. The ligation process requires energy and is the most efficient during the joining of sticky DNA ends, which are stabilized by hydrogen bonds of the joined strands (Fig. 3). Rys.3. Ligase activity scheme – joining of sticky ends Alkaline phosphatase – removes the phosphoric acid residue from the 5’ end of the plasmid, that had previously been cut by restriction enzymes thanks to which it makes self-ligation of the plasmid in the cloning process impossible (the ligase needs both groups at the end of the DNA strand – hydroxyl and phosphate – while after alkaline phosphatase only the hydroxyl group remains). Therefore, working with this enzyme we can prevent the closing of the plasmid-acceptor prior to cloning in the insert. As a result the insert is built-in correctly (it joins its own phosphate group at the 5’ end of the remaining hydroxyl group at the 3’ end of the vector), while the missing phosphodiester bond with a strand of a complementary group is then supplemented thanks to the mechanism or repairing DNA breaks in the host’s cell. Page 5 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Molecular cloning. Molecular cloning is the basic and necessary technique which allows for the construction and modification of genetic tools based on DNA molecules. New genetic constructs allow for the analysis of the function of genes, as well as expression of genes in bacteria or eukaryotic cells having a scientific-cognitive, industrial, and medical uses. Traditional molecular cloning is referred to as the isolation and amplification of a specific DNA fragment. Most such fragments are obtained through the digestion of an existing genetic material by restriction enzymes or its initial copying via the PCR technique. The course of basic molecular cloning (Fig.4): a) Preparation of the vector (the so-called acceptor) and the DNA fragment (the so-called insert) by:  Cutting and opening the vector with restriction enzymes and (optionally) digesting it by alkaline phosphatase,  Cutting out a DNA fragment from another vector (the so-called donor). The DNA insert may also be prepared from any DNA fragment using the polymerase chain reaction (PCR) multiplying in vitro a DNA sequence defined by starters. In the PCR, new sites of enzyme cutting may be added to the DNA fragment, which facilitates the cloning process. The most beneficial and unambiguous scheme of cloning a gen/insert of a given plasmid covers cloning with the use of two different restriction sites providing sticky ends present both in the insert and in the plasmid- recipient, enabling adding the insert in a specific orientation b) Ligation (joining) of isolated DNA fragments (vector and insert). c) Transformation of bacteria by the ligation product – via the thermal shock method or electroporation. d) Growing bacteria on agar plates with a specific antibiotic added, allowing for the growth of only the colonies of those bacteria that took a given plasmid with an inserted resistance gene. e) Collecting bacterial clones from the agar plates with the aim of isolating plasmids and identifying the properly recombinant plasmid through restrictive analysis and/or PCR method analysis (Fig. 3). Page 6 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Rys. 3. Transformation of bacteria obtained as a result of cloning with a plasmid as well as clone growth (source: e-biotechnologia.pl) Restriction analysis Restriction analysis mans the analysis of the size of DNA fragments created during plasmid digestion (obtained as a result of cloning) with restriction enzymes in order to confirm the proper cloning in of the new gene (insert). The aforementioned analysis requires the selection of the proper restriction enzymes, so that the combinations of the DNA fragment masses (created after the cleaving with those enzymes) would be unique for individual, assumed cloning results (meaning, unique for each of the plasmids which could theoretically be created during the ligation of the insert with the vector), The identification of DNA fragment masses crated during the cleaving with restrictive enzymes is performed by their electrophoretic separation in an agarose gel. The speed of the migration of DNA fragments (bands) in agarose is dependent on their mass – the longer fragments migrate slower. Page 7 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Fig. 4. Scheme of molecular cloning Practice Student:  Can analyze markers on plasmid maps,  Based on the available plasmid (donor and recipient) maps, design the digestion of the thread of plasmid DNA strand (the so-called donor), in order to isolate a specific DNA fragment (the so-called insert) and clone it into another plasmid vector (the so-called acceptor),  Recognize plasmid vectors, that are a result of cloning,  Calculate masses of DNA fragments, that are the results of a given cloning procedure,  Analyze the results of electrophoresis of DNA fragments created after the digestion of plasmid vectors with selected restriction enzymes. Remarks to exercises 1-3: Answers must be entered onto the worksheets. On the plasmid maps:  masses (lengths) of plasmids are specified in base pairs (bp),  value 1bp means the beginning of a plasmid, while the value placed below the name (e.g. 3782 in the case of plasmid pUC-OVA-GFP) means the end of the plasmid,  the beginning and end of the plasmid is assumed to be at “twelve o’clock”,  the burgundy color of the name of the enzyme means a single cleave in a given plasmid (one digestion site), black color of the enzyme means a double or multiple cleave, Page 8 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24  a marking of for example PsI (582) is a location of the recognized DNA sequence cleaved by the enzyme PsI – cleaving site (582) is an arbitrary number of base pairs counted from the beginning of the plasmid Exercise no 1 Planning a cloning strategy for DNA fragment DNA OVA-GFP (sample fusion protein Ovalbumin- Green Fluorescent Protein) from the plasmid pUC OVA-GFP (p. 10) into the expression plasmid pCMV (p, 11). Two options must be considered – the insert is built into the new plasmid vector at two different orientations (sub-point a and b) Please provide the different possible restriction enzyme combinations (from the included plasmid maps) that will allow the proper cloning of the aforementioned DNA fragment: a) so that the orientation of the OVA GFP gene would align with the orientation of the CMV promotor, Combination I PstI / XmaI Combination II ………/………. Combination III ………/………. Combination IV ………/………. Combination V ………/………. ………………………………………………………………………………………………………………… ………………………………………………………………………………………………………………… ………………………………………………………………………………………………………………… b) so that the orientation of the gene would be opposite to the orientation of the CMV promotor, Combination I EcoRI / BamHI Combination II ………/………. Combination III ………/………. Combination IV ………/………. Combination V ………/………. c) How would it be possible to clone in an OVA-GFP insert using only one restriction enzyme. Propose which enzyme may be used in this case. ………………………………………………………………………………………………………………. What can we connect such a method of cloning with? What must we remember when analyzing new plasmids constructed in this way? Page 9 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 ………………………………………………………………………………………………………………… ………………………………………………………………………………………………………………… ………………………………………………………………………………………………………………… How many theoretical combinations of vectors can be created using the above-mentioned cloning (extra-credit question)………………….................. d) If the restriction sites before and after the insert were not available what cloning strategy should be suggested? How would it be possible to obtain the DNA fragment in question (insert) and add the missing sites? …………………………………………………………………………………………………………………. Exercise no 2 Restriction analysis of cloning products. We assume that for the cloning described in exercise no. 1, we used the HindIII enzyme a) Based on the maps of newly constructed plasmids (p.12) with the provided restriction enzymes identify on a virtual agarose gel (p. 16) clone (A, B, C, D, or E) in which the OVA-GFP insert has the following orientation:  same as the CMV promoter - clone ……………  opposite to the CMV promoter - clone …………………. b) What must we remember when selecting enzymes for restriction analysis? ………………………………………………………………………………………………………… …………………………………………………………………………………………………………… Exercise no. 3 Fill in the 3 tables below each of the plasmid maps (pp. 13-15) entering in fragment masses in bp. Fragment mass is determined by calculating the difference in distance (in base pairs) between sites of cleaving with a given restriction enzyme. Pay attention to the method of calculating fragment mass when it contains a beginning and an end of pair numeration. Page 10 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 PLASMID MAPS (for exercise 1) PLASMID pUC OVA-GFP donor of the OVA-GFP fragment (insert) Page 11 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 PLASMID MAPS (for exercise 1) CMV promoter PLASMID OVA-GFP fragment (insert) acceptor Page 12 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 PLASMID MAPS (for exercise 2) PLASMIDS theoretically developed as a result of cloning through the HindIII restriction site Closed insert Double closed insert Double symmetrically closed insert Why is it impossible to maintain closed inserts in the host? (extra credit question) ……………………………………………………………………………………………… ………………………………………………………………………………………………. Double closed plasmid-acceptor Double symmetrically closed plasmid-acceptor Page 13 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 PLASMID MAPS (for exercise 3) PLASMIDS developed as a result of cloning through HindIII restriction sites which formed real clones Below is the expected clone, in which the insert is cloned into the vector-acceptor in line with the CMV promoter orientation. Subsequent pages show a non-functional vector and a vector without an insert. Expected vector, in line with the orientation of the insert and promoter Plasmid restriction analysis Calculate and enter in the masses (bp) of fragments which may be created as a result of a cleaving of the above plasmid with the following restriction enzymes. BamHI NcoI HindIII Page 14 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 PLASMID MAPS (for exercise 3) PLASMIDS develop as a result of cloning through the HindIII restriction site which formed real clones. Non-functional vector with insert in an orientation opposite the CMV promoter Plasmid restriction analysis Calculate and enter in the masses (bp) of fragments which may be created as a result of cleaving the above plasmid with the following restriction enzymes. BamHI NcoI HindIII Page 15 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 PLASMID MAPS (for exercise 3) PLASMIDS developed as a result of cloning through the HindIII restriction site that formed real clones Closed vector without an insert. The product of self-ligation. Treating the vector-acceptor with phosphatase should eliminate vector self-ligation. Nevertheless, this is a possible, unwanted product. Plasmid restriction analysis Calculate and enter in the masses (bp) of fragments which may be created as a result of the above plasmid with the following restriction enzymes. BamHI NcoI HindIII Page 16 of 18  WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Restriction digestion of plasmids isolated from 5 host clones (A-E) was conducted. During DNA electrophoresis DNA fragments formed during digestion whose mass corresponds to the distances between recognition sequences recognized by restriction enzymes were separated. Restriction Analysis of plasmids obtained after cloning the OVA-GFP fragment into the pCMV plasmid M – size marker (number of base pairs) On the agarose gel there are fragments of plasmids separated by electrophoresis and digested with restriction enzymes BamHl, Ncol, and HindIII. The analyzed plasmids were isolated from 5 randomly collected host clones A, B, C, D, and E Based on restriction analysis indicate which of the host clones contains the expected pCMV OVA-GFP plasmid Clone………….. Insert with an opposite orientation Clone………….. Does not contain an insert Clone…………. Page 17 of 18 WORKSHEET 4- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2023/24 Page 18 of 18