Molecular Biology Restriction enzymes

Restriction enzymes Endonuclease Cleaves internal phosphodiester linkages Recognize specific double stranded DNA sequences Different endonucleases recognize different sequences Recognize palindrome sequences

5’-G G A T C C-3’ 3’-C C T A G G-5’ Palindromes The same sequence is read in the 5’ » 3’ direction on both strands 5’-G G A T C C-3’ 3’-C C T A G G-5’

5’-GGATCC-3’ 3’-CCTAGG-5’ Palindromes The base at one position determines the corresponding base at the same position on the other strand 5’-GGATCC-3’ 3’-CCTAGG-5’

Palindromes CAC_ _ _ GT _ TA _ AT _ _ AT _ _ CACGTC GTATAC ATATATAT Complete the following palindromes CAC_ _ _ GT _ TA _ AT _ _ AT _ _ CACGTC GTATAC ATATATAT

Palindromes Problem How many palindromes of 4 bases are possible? N1N2N3N4 Position 1: 4 choices (A, G, C, or T) Position 2: 4 choices (A, G, C, or T) Position 3: 1 choice (determined by position 2) Position 4: 1 choice (determined by position 1) Therefore: 4 X 4 X 1 X 1 =16

How many palindromes does the enzyme AccI recognize? Problem How many palindromes does the enzyme AccI recognize? GTMKAC (M= A or C; K = G or T) 1 X 1 X 2 X 1 X 1 = 2

Frequency of Occurrence Probability of finding a given palindrome Depends on the base distribution Ex. 50% G or C or 1/4 for each base What is the probability of occurrence of GGATCC? 1/46 ou 1 divided by (0.25)-6 = 1/4096 How many times would this enzyme be expected to cut in a 10 kb genome? 1/4096 X 10000 = 2.44 Round off to nearest whole number. Therefore 2X

Frequency of Occurrence Problem What is the probability of finding any 4 base palindrome in a genome which is 50% G or C Number of different sequences of 4 bases 44 = 256 Number of different palindromes of 4 bases 4 X 4 X 1 X 1 = 16 Probability of finding a 4 base palindrome 16/256 = 1/16

Frequency of Occurrence Unequal distribution of bases Determine the sequence of each possible palindrome Independently determine probability of each palindrome according to base distribution Determine the sum of probabilities Ex. AccI cuts GTMKAC (M= A or C; K = G or T) Base distribution: 70% G/C, 30% A/T 2 palindromes possible GTATAC: (0.35)(0.15)(0.15)(0.15)(0.15)(0.35) = 1/16125 GTCGAC: (0.35)(0.15)(0.35)(0.35)(0.15)(0.35) = 1/2962 Probability: 1/2502

5’-G 3’-C C T A G G A T C C-3’ G-5’ Cleavage The same phosphodiester linkages are cleaved on both strands! 5’-G 3’-C C T A G G A T C C-3’ G-5’

Different ends are generated 3’-C C T G A A G T C C-3’ G-5’ Blunt ends

Different ends are generated 3’-C C T A G G A T C C-3’ G-5’ 5’ overhangs

Different ends are generated 3’-C 5’-G G A T C C-3’ C T A G G-5’ 3’ overhangs

Compatibility of ends Blunt ends HO P OH O P Compatible

Compatibility of ends Overhangs HO P OH HO P O Incompatible

Compatibility of ends Annealing Compatible Overhangs P-CTAG HO GATC-P OH Annealing GATC-P O P-CTAG O Compatible

Compatibility of ends Annealing Incompatible Overhangs GATC-P HO OH P-TCCA HO GATC-P OH Annealing GATC-P OH P-TCCA HO Incompatible

Restriction Maps

Compaible ends Which restriction sites in List 1 would generate compatible ends with those generated from List 2? List 1 GA▼TATC AAAT ▼ TT CGC ▼ CGC A ▼ CGCGT List 2 CCAT ▼ GGC CAC ▼ GTG CCGC ▼ GG CCATAT ▼ ATGG 2+A & 2+D

Restriction maps Determining the positions of restriction sites Linear DNA maps Circular DNA maps (plasmids) Maps of inserts within vectors Mapping genomes Southern analysis

Approach Determine whether the DNA has digested Is the digestion complete or partial? How many cuts? Determine the relative positions

Is the DNA digested? Compare to the undigested control Ladder Control Which samples were not digested? 1 and 4 Which samples were digested? 2 and 3 1 2 3 4

Is the digestion complete? Complete digestion All the DNA molecules are cleaved at all the possible sites Partial digestion A fraction of the molecules are not digested Partial undigested A fraction of the molecules were digested, but not at all the possible sites

Complete digestion Digestion

Partial digestion: Partial undigested Non digested Digestion

Partial digestion partial Digestion

Is the digestion complete or partial? Ladder Control 1 2 3 4 Compare to control Verify the intensity of the bands Verify the sizes

Determine the relative positions How many cuts? Number of sites Circular DNA = number of bands Linear DNA = Number of bands – 1 Determine the relative positions The fragment sizes represent the distances between the sites

Linear DNA maps HindIII HindIII + SalI Enzyme Fragments (Kb) HindIII 3 and 4 SalI 2 and 5 HindIII + SalI 2 and 3 7.0 3.0 4.0 HindIII HindIII + SalI 2.0 3.0

Circular DNA maps (plasmids) Enzyme Fragments (Kb) BamHI 2, 3 and 5 HindIII 1 and 9 BamHI + HindIII 1, 1.5, 2, 2.5 and 3 3.0 2.0 1.0 1.5 2.5 7.0 10.0 1.0 9.0 10.0

Recombinant plasmids MCS X Digested with X Vector

Recombinant plasmid + Insert X Recombinant Vector + insert

Determining restriction map of a DNA insert Step 1: Determining Insertion Site + Insert X Cut with X

Insertion maps Total size Insert size Insertion site 7.7Kb Generates 2 fragments one of which is the size of the vector XbaI Enzyme Fragments BamHI 7.7Kb EcoRI 1.0, 3.0, 3.7Kb PstI 2.0 and 5.7 XbaI 2.7 and 5.0

Determining Relative Orientation Insertion site delimits Right & Left Ex. If Pst is the insertion site: Bam is to the left. If Xba is the insertion site, then Bam is to the right

Determining Relative Orientation X A X Orientation 2 A X A X

Relative mapping Sites to map Enzyme Fragments Total cuts Sites in vector Sites in insert BamHI 7.7Kb 1 EcoRI 1.0, 3.0, 3.7Kb 3 2 PstI 2.0 and 5.7 XbaI 2.7 and 5.0 Insertion site

Map of PstI : 2 and 5.7Kb 5.7 Kb 2.0 Kb 5.0

Map of EcoRI: 1, 3 and 3.7Kb P 3.7 1.0 3.0 1.0 1.0 3.0

Restriction analysis of large DNA Purpose Find the position of a sequence in a large DNA fragment Find similar sequences in other organisms Analyze non-sequenced DNA

Digestion of Large DNA Number of fragments makes analysis impossible

Southern Analysis Separate DNA fragments according to size Denature Hybridize with single stranded probe representing region of interest Probe allows visualization of only the region of interest

Southern Probe Agarose gel Hybridize to probe

Problem What fragment sizes would be observed with probes 1 and 2 following sigle digests with each enzyme?

Solution Enzyme Probe 1 Probe 2 C 2.5 & 11kb 11kb K >11kb X >9.8 & 10.2kb >9.8kb