DNA CLONING DNA CLONING Dr.Sarookhani Dr.Sarookhani / / - PDF document

�� / �� / ���� DNA CLONING DNA CLONING Dr.Sarookhani Dr.Sarookhani �

�� / �� / ���� Dr.Sarookhani Dr.Sarookhani �

�� / �� / ���� Dr.Sarookhani Dr.Sarookhani �

�� / �� / ���� Dr.Sarookhani Dr.Sarookhani �

�� / �� / ���� WAYS OF GENERATING WAYS OF GENERATING DNA DNA FRAGMENTS FRAGMENTS 1. 1. Non Non- -specific specific generation generation of of truly truly random random fragments fragments (by (by mechanical mechanical shearing shearing or or digestion digestion with with non non- - specific specific nucleases) nucleases) 2. 2. Through Through reverse reverse transcription transcription of of mRNA mRNA into into DNA DNA 3. 3. Highly Highly specific specific amplification amplification of of a a chosen chosen piece piece of of DNA by DNA by PCR PCR 4. 4. The The use use of of synthetic synthetic DNA DNA 5. 5. Restriction Restriction endonucleases endonucleases digestion digestion Dr.Sarookhani Dr.Sarookhani �

�� / �� / ���� ENZYMES IN CLONING ENZYMES IN CLONING Cutting and joining DNA Cutting and joining DNA Dr.Sarookhani Dr.Sarookhani �

�� / �� / ���� RESTRICTION ENDONUCLEASES RESTRICTION ENDONUCLEASES • • Enzymes Enzymes • • Recognizes Recognizes a a short, short, symmetrical symmetrical DNA DNA sequence sequence • • Hydrolyzes/cuts Hydrolyzes/cuts the the DNA DNA backbone backbone in in each each strand strand – – Specific Specific site site within within that that sequence sequence – Foreign Foreign DNA DNA is is degraded degraded into into short short fragments fragments – – Finger Finger printing printing – Dr.Sarookhani Dr.Sarookhani �

�� / �� / ���� RESTRICTION ENDONUCLEASES RESTRICTION ENDONUCLEASES 2. 2. Part Part of of the the restriction restriction- -modification modification defense defense mechanism mechanism against against foreign foreign DNA DNA 3. 3. Basic Basic tools tools of of gene gene cloning cloning Dr.Sarookhani Dr.Sarookhani �

�� / �� / ���� RESTRICTION ENDONUCLEASES RESTRICTION ENDONUCLEASES 3 types types Type I Type I Type II Type II Type III Type III Dr.Sarookhani Dr.Sarookhani �

�� / �� / ���� TYPE II REs TYPE II REs • • commonly commonly used used in in cloning cloning • • recognize recognize and and cut cut within within (or (or immediately immediately adjacent adjacent to) to) specific specific target target sequences sequences – – generate generate specific specific fragments fragments • • a a small small number number – cut cut the the DNA DNA at at a a defined defined distance distance (usually (usually only only a a – few few bases) bases) away away from from the the recognition recognition site site – limited limited applications applications – • • requirement Mg 2 2+ + requirement: : Mg Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� CHARACTERIZATION AND IDENTIFICATION CHARACTERIZATION AND IDENTIFICATION 1. 1. The name of the organism from which The name of the organism from which they are obtained they are obtained 2. 2. Write in Write in italics italics • • The first letter of the genus The first letter of the genus • • The first two letters of the species The first two letters of the species name name 3. 3. A suffix indicating the specific enzyme A suffix indicating the specific enzyme from that species from that species Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� CHARACTERIZATION AND CHARACTERIZATION AND IDENTIFICATION IDENTIFICATION • • Example: Example: – Pst – Pst I from I from Providencia stuartii Providencia stuartii – Hae Hae I, I, Hae Hae II and II and Hae Hae III, three different enzymes, III, three different enzymes, – with different specificities from with different specificities from Haemophilus Haemophilus aegyptius aegyptius Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� THE PRODUCT OF REs DIGESTION THE PRODUCT OF REs DIGESTION 1. 1. Products Products with with protruding protruding ends ends known known as as cohesive cohesive or or ‘ ‘ sticky sticky ’ ’ ends ends • • Fragments Fragments with with unpaired unpaired single single- -stranded stranded sequences either sequences either at at the the 5 5 ’ ’ or or 3 3 ’ ’ ends ends Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� THE PRODUCT OF REs DIGESTION THE PRODUCT OF REs DIGESTION 5’-G-3’ 5’-AATTC-3’ 5’-GAATTC-3’ + (a) 3’-CTTAA-5’ 3’-G-5’ 3’-CTTAAG-5’ Eco RI 5’ overhang forms cohesive ends (b) 5’-CTGCAG-3’ 5’-CTGCA-3’ 5’-G-3’ + 3’-GACGTC-5’ 3’-G-5’ 3’-ACGTC-5’ Pst I 3’ overhang forms cohesive ends Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� THE PRODUCT OF REs DIGESTION THE PRODUCT OF REs DIGESTION 2 2. . Products with blunt ends Products with blunt ends Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� • • Example Example 5’-CCCGGG-3’ 5’-CCC-3’ 5’-GGG-3’ + 3’-GGGCCC-5’ 3’-GGG-5’ 3’-CCC-5’ Sma I • • Advantage: Advantage : they they can can be be joined joined to to any any other other blunt- blunt -ended ended fragment fragment • • Disadvantage: Disadvantage : less less efficiently efficiently ligated ligated Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� Enzymes Enzymes Recognition site Recognition site Number of Number of Ends generated Ends generated Source Source bases bases Eco RI Eco RI 5 ’ 5 ’ sticky sticky Escherichia coli Escherichia coli RY RY13 13 G/AATTC G/AATTC 6 6 Bam Bam HI HI 5 5 ’ ’ sticky sticky Bacillus Bacillus G/GATCC G/GATCC 6 6 amyloliquefaciens H amyloliquefaciens H Bgl Bgl II II A/GATCT A/GATCT 6 6 5 5 ’ ’ sticky sticky Bacillus globigii Bacillus globigii Pst Pst I I 3 ’ 3 ’ sticky sticky Providencia stuartii Providencia stuartii CTGCA/G CTGCA/G 6 6 Xma Xma I I C/CCGGG C/CCGGG 6 6 5 ’ 5 ’ sticky sticky Xanthomonas Xanthomonas malvacearum malvacearum Sma I Sma I Serratia marcescens Serratia marcescens CCC/GGG CCC/GGG 6 6 blunt blunt Sau3 Sau 3A A /GATC /GATC 4 4 5 5 ’ ’ sticky sticky Staphylococcus aureus Staphylococcus aureus 3 3A A Alu I Alu I AG/CT AG/CT 4 4 blunt blunt Arthrobacter luteus Arthrobacter luteus Not I Not I GC/GGCCGC GC/GGCCGC 8 8 5 5 ’ ’ sticky sticky Nocardia otitidis- Nocardia otitidis - caviarum caviarum Pac Pac I I TTAAT/TAA TTAAT/TAA 8 8 3 3 ’ ’ sticky sticky Pseudomonas Pseudomonas alcaligenes alcaligenes Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� INSERT VECTOR CIRCULAR MONOMERS LINEAR DIMERS (OR HIGHER MULTIMERS CIRCULAR DIMERS RECOMBINANT PLASMID SOME POTENTIAL PRODUCTS OF LIGATION Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� OTHER ENZYMES IN CLONING OTHER ENZYMES IN CLONING • • Nucleases Nucleases • • Ligases Ligases • Phosphatase • Phosphatase and and Kinases Kinases • • DNA DNA synthesizing synthesizing enzymes enzymes Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� NUCLEASES NUCLEASES Cutting/degrading DNA Cutting/degrading DNA ENDONUCLEASES ENDONUCLEASES EXONUCLEASES EXONUCLEASES MULTIFUNCTIONAL NUCLEASES MULTIFUNCTIONAL NUCLEASES Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� LIGATION LIGATION • • The The next next stage stage in in gene gene cloning cloning – joining – joining the the DNA DNA fragment fragment to to a a vector vector molecule molecule • • DNA DNA ligase ligase Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� p OH OH p p OH p OH recombinant vector molecule LIGATION Dr.Sarookhani Dr.Sarookhani ��

�� / �� / ���� PHOSPHATASES AND KINASES PHOSPHATASES AND KINASES • • Removing or adding respectively phosphate groups Removing or adding respectively phosphate groups • • Examples: Examples: – alkaline phosphatase: removes the alkaline phosphatase: removes the 5 5 ’ ’ terminal terminal – phosphate from a DNA molecule leaving an OH phosphate from a DNA molecule leaving an OH group group – polynucleotide kinase: adds a phosphate group polynucleotide kinase: adds a phosphate group – to a free 5 to a free 5 ’ ’ - -terminus terminus • • reverses the effect of alkaline phosphatase reverses the effect of alkaline phosphatase Dr.Sarookhani Dr.Sarookhani ��