Transformation Insertion of DNA of interest Amplification Amplified segment of DNA can be purified from bacteria in sufficient quantity and quality for : DNA Sequence. Understand relatedness of genes and organism. Diagnosis and treatment of genetic disease. Modify DNA sequence. Introduce mutation in the gene to study the gene function. Transformation into desired organism. Create genetic knock-outs (delete the gene from organism). For production of protein. Regulation of gene expression. 1
for DNA Manipulations Nuclease An enzyme that degrades a nucleic acid molecule. Exonuclease vs Endonuclease / Sequence specific vs non-specific Ligase An enzyme that synthesizes phosphodiester bonds as part of DNA replication, repair and recombination processes. End-modification enzyme: An enzyme that alters the chemical structure at the end of a DNA molecule. DNA Polymerase An enzymes that synthesize new polynucleotides complementary to an existing DNA. Restriction (Site-Specific Endonuclease) that recognize and cleave dsdna in a highly sequence specific manner. 2
Restriction (Site-Specific Endonuclease) that recognize and cleave dsdna in a highly sequence specific manner. Generally recognize an inverted repeat sequence 4, 6, or 8 base pairs in length [occurrences average every 4 4 (256bp), 4 6 (4kbp), or 4 8 (65kbp)] Restriction (Site-Specific Endonuclease) that recognize and cleave dsdna in a highly sequence specific manner. Generally recognize an inverted repeat sequence 4, 6, or 8 base pairs in length [occurrences average every 4 4 (256bp), 4 6 (4kbp), or 4 8 (65kbp)] Cleavage occurs to leave blunt ends, 3 or 5 over-hanging ends. Hind III 5 -- AAGCTT-- 3 3 - TTCGAA-- 5 Pst I 5 -- CTGCAG-- 3 3 - GACGTC-- 5 Eco RV 5 -- GATATC-- 3 3 - CTATAG-- 5 5 - A 3 5 AGCTT-- 3 5 - CTGCA 3 G-- 3 5 - GAT 3 5 ATC-- 3 3 - TTCGA 5 3 A-- 5 3 - G 5 ACGTC-- 5 3 - CTA 5 3 TAG-- 5 3
Restriction (Site-Specific Endonuclease) that recognize and cleave dsdna in a highly sequence specific manner. Generally recognize an inverted repeat sequence 4, 6, or 8 base pairs in length [occurrences average every 4 4 (256bp), 4 6 (4kbp), or 4 8 (65kbp)] Cleavage occurs to leave blunt ends, 3 or 5 over-hanging ends. Leaves 5 phosphate and 3 OH. Agarose Gel Electrophoresis 4
Agarose Gel Electrophoresis Can resolve DNA ~300 bp to ~20 kb. Ethedium bromide staining is directly proportional to the length of DNA. Separation is based on linear DNA molecule. Circular and Supercoil DNA migrate differently. Agarose Gel Electrophoresis Can resolve DNA ~300 bp to ~20 kb. Ethedium bromide staining is directly proportional to the length of DNA. Separation is based on linear DNA molecule. Circular and Supercoil DNA migrate differently. Phage Lambda DNA Digested with BstEII or HindIII, visualized by ethidium bromide staining of agarose gel. 5
PolyAcrylamide Gel Electrophoresis (PAGE) Can resolve DNA ~30 base to ~3000 base. Restriction (Site-Specific Endonuclease) Generation of Restriction map. Analysis 6.2 kb linear DNA of unknown sequence. Use multiple restriction enzymes to deduce the restriction sites on the DNA fragment. 6
DNase I (Non-Sequence Specific Endonuclease) that recognize and cleave dsdna in a non-sequence specific manner (random). Degree of cleavage depends on amount of enzyme and time of incubation. SI Nuclease (Single-strand Endonuclease) Degrades single-stranded DNA or RNA polynucleotides, including single-stranded regions in predominantly double-stranded molecules, but has no effect on doublestranded DNA or on DNA-RNA hybrids. 7
Mung Bean Nuclease (Single strand Exonuclease) Degrades single-stranded DNA or RNA polynucleotide. Act on either 5 or 3 end. DNA Ligase An enzyme that synthesizes phosphodiester bonds as part of DNA replication, repair and recombination processes. Requires: Double stranded DNA PO 4 group on 5 end of DNA. OH on 3 end of DNA. ATP molecule Overhangs must be compatible for efficient ligation to occur. 8
DNA Ligase An enzyme that synthesizes phosphodiester bonds as part of DNA replication, repair and recombination processes. Requires: Double stranded DNA PO 4 group on 5 end of DNA. OH on 3 end of DNA. ATP molecule Overhangs must be compatible for efficient ligation to occur. End-modification enzymes: Terminal deoxynucleotidyl transferase. Added nucleotides one after the other to the 3 terminus at a blunt end. 9
End-modification enzymes: Terminal deoxynucleotidyl transferase. Added nucleotides one after the other to the 3 terminus at a blunt end. T4 polynucleotide kinase An enzyme that adds phosphate groups to the 5 - OH ends of DNA molecules. Useful for ligating oligonucleotides. End-modification enzymes: Terminal deoxynucleotidyl transferase. Added nucleotides one after the other to the 3 terminus at a blunt end. T4 polynucleotide kinase An enzyme that adds phosphate groups to the 5 - OH ends of DNA molecules. Alkaline phosphatase An enzyme that removes phosphate groups from the 5 ends of DNA molecules and thus prevent ligation. 10
DNA Polymerase An enzymes that synthesize new polynucleotides complementary to an existing DNA. Requires a primer in order to initiate the synthesis of a new polynucleotide. Short DNA or RNA (oligonucleotide) up ~ 100 nts can be synthesized chemically Custom DNA and RNA are available for purchase online. DNA Polymerase An enzymes that synthesize new polynucleotides complementary to an existing DNA. Requires a primer in order to initiate the synthesis of a new polynucleotide. E.coli DNA pol I has exonuclease activities: 3 -> 5 : Removes incorrect nucleotide incorporated. 5 ->3 : Remove nucleotides from 5 end of polynucleotides Klenow polymerase: E.coli DNA pol I which lacks 5 ->3 exonuclease activity. 11
DNA Polymerase An enzymes that synthesize new polynucleotides complementary to an existing DNA. Requires a primer in order to initiate the synthesis of a new polynucleotide. E.coli DNA pol I has exonuclease activities: 3 -> 5 : Removes incorrect nucleotide incorporated. 5 ->3 : Remove nucleotides from 5 end of polynucleotides Klenow polymerase: E.coli DNA pol I which lacks 5 ->3 exonuclease activity. Molecular Cloning 1. Isolation of DNA. 2. Ligating the DNA fragment into a vector. 3. Transformation of a host cell with the recombinant DNA. 4. Selection of host cells containing the recombinant DNA. 5. Screening cells for those with recombinant DNA or producing a protein of interest. 12
Molecular Cloning Ligating the DNA fragment into a vector. Cloning vector needs to have the following characteristics: 1. Have an origin of replication so that the DNA can be replicated within a host cell. 2. Have one or more selectable markers for determining whether the cloning vehicle has been transferred into cells and to indicate whether the foreign DNA has been inserted into the vector. 3. Generally have several unique restriction sites for cloning a DNA fragment (called a multiple cloning site, or MCS ) so that the vector will be cut with desired restriction enzyme. Molecular Cloning 13
Molecular Cloning Ligating the DNA fragment into a vector. Cloning vector needs to have the following characteristics: 1. Have an origin of replication so that the DNA can be replicated within a host cell. 2. Have one or more selectable markers for determining whether the cloning vehicle has been transferred into cells and to indicate whether the foreign DNA has been inserted into the vector. 3. Generally have several unique restriction sites for cloning a DNA fragment (called a multiple cloning site, or MCS ) so that the vector will be cut with desired restriction enzyme. Molecular Cloning Ligating the DNA fragment into a vector. Cloning vector needs to have the following characteristics: 1. Have an origin of replication so that the DNA can be replicated within a host cell. 2. Have one or more selectable markers for determining whether the cloning vehicle has been transferred into cells and to indicate whether the foreign DNA has been inserted into the vector. 3. Generally have several unique restriction sites for cloning a DNA fragment (called a multiple cloning site, or MCS ) so that the vector will be cut with desired restriction enzyme. 14
Molecular Cloning Ligating the DNA fragment into a vector. Molecular Cloning Troubleshooting: High background: vector ligating without insert. a) Use two different restriction enzymes. b) Dephosphorylate the vector with phosphatase. Insert ligate in opposite orientation. Use two different restriction enzymes. Alter one end of restriction site (by phosphatase, Mungbean Nuclease, DNA pol) to achive directional cloning. Multiple insert in single vector. Reduce amount of insert. Dephosphorylate the insert. 15