BT-620 Lecture 1 PDF

Basics of Gene Cloning Nucleic Acid Isolation & Cloning Enzymes Lecture 1 BT-620  What is DNA cloning? In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA mo...

Basics of Gene Cloning Nucleic Acid Isolation & Cloning Enzymes Lecture 1 BT-620  What is DNA cloning? In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes  Bacterium Cell containing gene Why Clone DNA? Gene inserted into of interest plasmid A particular gene can be isolated Bacterial Plasmid and its function determined chromosome Gene of Recombinant interest DNA (plasmid) DNA of chromosome Control sequences of DNA can be Plasmid put into bacterial cell identified & analyzed Recombinant bacterium Protein/enzyme/RNA function can Host cell grown in culture to form a clone of cells be investigated containing the “cloned” gene of interest Mutations can be identified, e.g. Gene of interest Protein expressed by gene of interest gene defects related to specific Copies of gene Protein harvested diseases Basic research and Basic various applications Basic research research Organisms can be „engineered‟ for on gene on protein specific purposes, e.g. insulin production, insect resistance, etc. The concept of “Pharm”animals Basic Research Gene for pest resistance inserted Gene used to alter bacteria for cleaning Protein dissolves blood clots in heart Human growth hor- mone treats stunted into plants up toxic waste attack therapy growth  Recombinant DNA Products Human Insulin Human growth factor Hepatitis B virus vaccine Streptokinase Recombinant antibodies Recombinant Enzymes Safflower  Regulations to monitor rDNA International and National rules that regulate rDNA organisms Special permissions, facilities and supervisory bodies to oversee & regulate the use rDNA organisms and products 1. DNA is extracted from blood/tissue 2. PCR/RE digestion of DNA & vector 3. Ligation in the vector How is DNA cloned ? 6. Expression in a suitable host 5. Screening for positives 4. Transformation into a host Isolation of Nucleic Acids  Genomic DNA Useful for obtaining entire coding region of prokaryotic organisms cDNA Obtaining coding region of eukaryotes  Genomic DNA Genomic DNA is the entire DNA content of an organism Suitable for determining genomic DNA sequence: Looking at regulatory elements or entire genes (exons & introns) Requires chromosomal DNA isolation.  Genomic DNA Isolation A source of cells Lysis by buffered solution containing detergent Digestion of cellular proteins using Proteinase K Removal of these proteins by either Phenol, Cholroform or salt Precipitation of Genomic DNA by Isopropanol Removal of any excess salts by Ethanol wash Resuspension in a suitable buffer  cDNA (Complementary DNA) Why is it needed? What does this involve? Purification of mRNA Synthesis of first strand (cDNA) Synthesis of second strand Cloning Transcription Exons Exons Exons Exons 1 2 3 4 DNA Introns Introns Introns 1 2 3 4 AAAA mRNA 1 2 3 4 AAAA Intron splicing 1 2 3 4 AAAA Processed mRNA Purification of RNA AAAA AAAA AAAA AAAA AAAA AAAA Cell Lysis Protein pptn. Removal/ pptn of DNA AAAA TTTT AAAA TTTT TTTT AAAA TTTT TTTT AAAA AAAA AAAA TTTT TTTT TTTT AAAA TTTT AAAA TTTT AAAA TTTT TTTT = + AAAA TTTT AAAA TTTT TTTT AAAA TTTT TTTT AAAA TTTT TTTT tRNA, rRNA AAAA Total RNA TTTT Oligo dT sepharose t, r& m mRNA  Purification of RNA A cell expressing the required protein A clean environment. Sterile glassware, plasticware free of nucleases, RNA inhibitors Purification involves the lysis of the cell Removal of contaminating proteins by Guanidinium isothiocyanate and subsequently chloroform extraction Removal of contaminating DNA by extraction with acid phenol and Rnase free DNase Total RNA prepared by such a method may now be used to purify mRNA The fact that eukaryotic mRNA has a poly A tail is exploited. A oligo dT sequence attached to sepharose / magnetic particles is used to fish out the cellular mRNA This mRNA can now be used to prepare cDNA  C G A T A G T C G A A T T C C A A T C C C GA A A A RNA Pol G C T A T C A G C T T C C G G T T A G G G C T T T T 5’ G A U A G U C G A A G G C C A A U C C CRev.G A A A A CRNA 3’ mRNA Pol Transcr. 3’ G CC T A T C A G C TT T CC C GG G T TT AA GG G GG C T T T TT 5’ cDNA 5’ 3’ 3’ AAAA TTTT cDNA-mRNA hybrid 5’  mRNA to cDNA 5’ 3’ 3’ AAAA 5’ TTTT ds cDNA Rnase H DNA Polymerase I DNA Ligase 3’ endonuclease 5’ to 3’ polymerase activity 3’ to 5’ exonuclease activity  Commonly used Vectors Plasmid Vectors: Circular, ds DNA that is capable of independent replication. It is widely used in rDNA techniques Phage based vectors: These are suitable for cloning larger fragments of DNA. Plasmid DNA Isolation Plasmid isolation is based on the alkaline lysis method developed by Birnboim and Doly (1979) The initial method utilized phenol:chloroform, purifications and ethanol precipitation Current spin column based protocols depend upon DNA binding to positively charged matrices. Current spin column based protocols depend upon DNA binding to positively charged matrices such as silica. DNA Binding High Salt Wash Elution Cutting and pasting are two of the first skills children learn, and the tools they use are scissors and glue. Similarly, cutting DNA and pasting DNA fragments together typically are among the first techniques learned in the molecular biology lab and are fundamental to all recombinant DNA work. Common Terminology of Cloning Enzymes 1. Kinases 2. Phosphatases 3. Reverse transcriptase 4. Endonucleases 5. Exonucleases 6. Polymerase  Phosphatases & Kinases Phosphatases remove the phosphate group from nucleic acids or proteins. These enzymes are most active at alkaline pH - hence the name. In molecular biology phosphatases are used to remove the 5’ PO4 from a DNA strand The commonly used phosphatases are Bacterial Alkaline phosphatase (BAP) Calf Intestinal alkaline phosphatase (CIAP) Shrimp Alkaline phosphatase 5’ 5’ 5’ 5’ Bacterial alkaline Phosphatase (BAP) is the most active enzyme but also the most difficult to destroy after the dephosphorylation reaction CIAP is the most commonly used in Molecular Biology, though less active than BAP, it is easily destroyed by heating at 75˚C in the presence of EDTA. The amount of CIAP/ug DNA as well as the time has to be carefully selected. Shrimp Alkaline Phosphatase is derived from a cold-water shrimp and is easily destroyed by heat (65 ˚C for 15 minutes).  Kinase Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer of a phosphate from ATP to the 5' end of either DNA or RNA. It is a product of the T4 bacteriophage, and commercial preparations are usually products of the cloned phage gene expressed in E. coli. The enzymatic activity of PNK is utilized in two types of reactions: The "forward reaction" : PNK transfers the phosphate from ATP to the 5' end of a polynucleotide (DNA / RNA). The target nucleotide is lacking a 5' phosphate either because it has been dephosphorylated or has been synthesized chemically. 5’ HO- -OH 3’ ATP PNK ADP 5’ PO4 -OH 3’ The "exchange reaction“: Target DNA or RNA that has a 5' phosphate is incubated with an excess of ADP PNK will first transfer the phosphate from the nucleic acid onto an ADP, This forms ATP and leaves a dephosphorylated target. PNK will then perform a forward reaction and transfer a phosphate from ATP onto the target nucleic acid. 5’PO4 -OH 3’ ADP PNK ATP 5’ HO- -OH 3’ ATP PNK ADP 5’PO4 -OH 3’ In combination Phosphatase and Kinase may be used for Radiolabeling probes Phosphatase : Removing 5' phosphates from fragments of DNA prior to labeling with radioactive phosphate 5’ PO4 5’ PO4 Kinase: Radiolabeling oligonucleotides, usually with 32P, for use as hybridization probes. 5’ HO- -OH 3’ ATP32 PNK ADP 5’ P32 -OH 3’ NUCLEASES Nucleases are enzymes that break the chemical bonds, called phosphodiester bonds, that hold the nucleotides of DNA or RNA polymers together. Enzymes that cleave the phosphodiester bonds of DNA are called deoxyribonucleases, Enzymes that cleave the phosphodiester bonds of RNA are called ribonucleases The endonucleases cleave phosphodiester bonds of DNA or RNA at positions other than at the end of the polymer. The cutting reactions of the endonucleases produce fragments of DNA or RNA. Exonucleases are involved in trimming the ends of RNA and DNA polymers, cleaving the last phosphodiester bond in a chain. This cleavage results in the removal of a single nucleotide from the polymer. If the enzyme removes nucleotides from the 3′ end, it is referred to as a 3′ exonuclease. If cleavage is at the 5′ end, the enzyme is called a 5′ exonuclease.  MOLECULAR SCISSORS In 1962, Werner Arber, a Swiss biochemist, provided the first evidence for the existence of "molecular scissors" that could cut DNA. He showed that E. coli have an enzymatic immune system that recognizes and destroys foreign DNA, and modifies native DNA to prevent self-destruction. Arber and his associates called this group of DNA-cutting enzymes restriction endonucleases. They also proved that restriction endonucleases are specific in their snipping: a given enzyme will cut DNA only when it recognizes exactly the right sequence. Present day recombinant technology remains totally dependent on this ability to cut DNA specifically!! RE’s are classified according to based on their properties Type I Type II Type III  TYPE I ENZYMES: Multifunctional enzyme Complex multi subunit enzymes 3 different subunits Combination of Restriction-Modification activities Specific recognition sequence, but cutting is random could be 1000 bp from this sequence. Requirements for restriction Mg 2+, ATP and S- adenosyl methionine Methylation occurs at the host specificity site Not of use in Gene Cloning due to their random cleavage of DNA  TYPE III ENZYMES: Separate endonuclease and methylase enzymes with a subunit in common Two different subunits enzymes Mg 2+, ATP are required for restriction. Specific recognition sequence, recognize two separate non-palindromic Cutting occurs 24-26 bp from the recognition sequences. These enzymes show rotational symmetry Methylation occurs at the host specificity site  TYPE II ENZYMES: Separate endonuclease and methylase enzymes Single subunit enzymes Mg 2+ is required for restriction. Specific recognition sequence, that is palindromic (4-8 nucleotides in length) Cutting occurs at specific sequences within or close to the site of recognition. These enzymes show rotational symmetry Methylation occurs at the host specificity site Several of these enzymes are routinely used in Gene Cloning due to their specificity of cleavage. Type II RE recognize palindromic sequences: "MADAM, I'M ADAM” "ABLE WAS I ERE I SAW ELBA" "DENNIS SINNED" Bam HI 5‟-GGATCC-3‟ 3‟-CCTAGG-5‟ Eco RI 5‟ -GAATTC-3‟ 3‟-CTTAAG-5‟ Xma I 5‟-CCCGGG-3‟ 3‟-GGGCCC-5‟ They could be 4 base, 6 base or 8 base cutters  Nomenclature of RE‟s The first letter (in capitals) is the first alphabet of the genus name of the host organism,followed by the first two letters of the specific epithet. e.g Escherichia coli = Eco Haemophilus influenzae = Hin Strain or type identification is written as subscript e.g Ecok. When a particular host strain has several different RE’s these are identified by roman numerals such as Hind II & Hind III Restriction Enzymes are prefixed with “R” R.Hind III Modification enzymes are prefixed with “M” M.Hind III In practice, subscripts are written in a single line Where the context the prefix is omitted e.g Hind III, Eco RI etc.. Eco RI 5‟- - 3‟ CGATAGTCGAA TTCCAATCCCG 3‟- GCTATCTGCTT AAGGTTAGGGC - 5‟ AATTCCAATCCCG CGATAGACG GTTAGGGC GCTATCTGCTT AAG Generates protruding 5‟ termini. Ends can associate due to the hydrogen bonding between overlapping 5‟ termini. These are called Sticky or Cohesive ends  Pst I generates 3‟ overhangs 5‟- - 3‟ CGATAGTCCTG CAGATCCCG 3‟- GCTATCTGGAC GTCTTAGGGC - 5‟ 5‟- CGATAGACCTGCA -3‟ 3‟- GCTATCTGG - 5‟ 5‟ GAATCCCG - 3‟ 3‟ ACGTCTTAGGGC - 5‟ Sma I ATTAAGATCCC GGGTTCGAGAC TAATTCTAGGG CCCAAGCTCTG ATTAAGATCCC GGGTTCGAGAC TAATTCTAGGG CCCAAGCTCTG Enzymes may also generate flush ends or Blunt ends  Isoschizomers: Two enzymes that cut a single sequence ATTAAGATCCC GGGTTCGAGAC TAATTCTAGGG CCCAAGCTCTG Sma I Blunt ends ATTAAGATCCC GGGTTCGAGAC TAATTCTAGGG CCCAAGCTCTG Xma I CCGGGTTCGAGAC ATTAAGATC CAAGCTCTG TAATTCTAGGGCC Sticky Ends  Star Activity Several enzymes exhibit Star Activity or the relaxation of specificity of a restriction enzyme under certain conditions such as : 1. Low ionic strength 2. High enzyme concentration 3. High glycerol content 4. In the presence of divalent cation other than Mg2+ such as Co 2+, Cu 2+, Mn 2+ & Zn 2+ 5. High pH 6. The presence of organic solvents such as ethanol, DMSO & ethylene glycol. Eco RI 5‟- G AATTC -3‟ Eco RI* 5‟- N AATTN-3‟ 3‟- CTTAA G-5‟ 3‟- NTTAA N-5‟ Bam HI 5‟-G GATCC-3‟ Bam HI * N GATCC, G PuATCC G GNTCC 3‟-CCTAG G-5‟ NCTAG G CPyTAG G CCNAG G  Ligase or Molecular Glue ! The problem of DNA joining was solved by work carried out in the late 1960s. Several laboratories simultaneously discovered DNA ligase, an enzyme that catalyses the formation of a phosphodiester bond between two DNA chains (Weiss and Richardson, 1967; Zimmerman et al., 1967). DNA ligase enzymes require a free hydroxyl group at the 3‟-end of one DNA chain and a phosphate group at the 5‟-end of the other. The formation of a phosphodiester bond between these groups requires energy. In E. coli and other bacteria NAD+ serves this role, whereas in animal cells and bacteriophage ATP drives the reaction. DNA ligases are only able to join DNA molecules that are part of a double helix – they are unable to join two molecules of single- stranded DNA. Not long after Arber's discoveries, Arthur Kornberg utilized this pasting mechanism for DNA, using the enzyme ligase. Kornberg was trying to construct artificial viral DNA from viral fragments, but had been unable to make a biologically active molecule. Once he added ligase, however, he found that the enzyme made it possible to paste the ends of DNA molecules together. With ligase, the viral DNA he created formed a continuous loop, just as it did in the original virus. The artificial viral DNA was indeed biologically active – it could reproduce on its own – and Kornberg was hailed as having "made life in a test tube."  DNA Ligases ATP dependent DNA Ligase (Phage) NAD dependent DNA Ligase (E.coli) The commonly used Ligase is from T4 phage  ATP AMP PPi Ligase P P P P P P PP P P P PP OH P P P P PP PP A A T T C C A A T C C C G C GA T A G T C G G T T A G G G C G C T A T C A G C T T C C G P P P P P P P OH P P P P P P P P P P P P P P AMP How can RE + Ligases be used in Cloning? DNA Polymerase 5’ 3’ 3’ 5’ Extension 5’ 3’ 3’ 5’ 5’-3’ Extension (error) 5’ 3’ 3’ 5’ 3’-5’ Proofreading 5’ 3’ 3’ 5’ 5’ 3’ Extension 3’ 5’  DNA Polymerase DNA polymerase can add free nucleotides to only the 3’ end of the newly-forming strand. This results in elongation of the new strand in a 5’-3’ direction. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleotide onto only a pre-existing 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide. Primers consist of RNA and DNA bases with the first two bases always being RNA, and are synthesized by another enzyme called primase. Error correction is a property of some, but not all, DNA polymerases. This process corrects mistakes in newly-synthesized DNA. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA. The 3‘- 5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading).