Chapter_4___Cutting_and_joining_DNA_v2

Chapter_4___Cutting_and_joining_DNA_v2 -...

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Cutting and Joining DNA 4.1 Restriction endocleases ( key concept: cutting DNA with compatible ends ) 4.11 Specificity 4.12 Sticky and Blunt ends, Isoschizomers, Processing restriction fragments 4.2 Ligation ( key concept: optimizing one DNA ligated per vector ) 4.2.1 Optimizing ligation conditions 4.2.2 Preventing unwanted ligation: alkaline phosphatase and double digests 4.3 Modification of Restriction Fragment Ends ( key concept: creating compatible ends by modification ) 4.3.1 Trimming and filling, Linkers / Adaptors 4.3.2 Homopolymer tailing 4.4 Other ways of joining DNA molecules ( key concept: joining PCR product ends to a vector without restriction sites ) 4.4.1 TA cloning of PCR products 4.4.2 DNA topoisomerase
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Restriction endonucleases derive their name from “ host controlled restriction and modification Restriction of phage growth obtained from one bacterial strain into another bacterial strain. Host bacteria produces restriction endonuclease that cuts foreign phage DNA Host strain DNA is protected by methylation Type I restriction endonucleases produces non-specific, DNA breakage Commonly used enzymes are Type II restriction endonucleases which cut at specific sites very close to the recognition sequence Figure 4.1 Bacteriophage restriction 4.1 Restriction Endonucleases
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Type II restriction endonucleases are named with first letter of the genus and first two letters of the species name Length of the recognition site (affects the frequency of cutting DNA) 8 base recognition enzymes such as NotI are excellent multiple cloning site enzymes because they rarely cut the insert Frequency of cutting is not always predictable G+C content varies among species Certain combinations of bases are more uncommon than they should Host methylation of DNA Dam and Dcm methylases is present in most strains of laboratory bacteria, preventing activity of some restriction enyzmes Corresponding CpG methylase in many eukaryotes inhibits restriction enzyme activity 4.1 Restriction Endonucleases 4.1.1 Specificity Box 4.1 Examples of restriction endonucleases
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4.1 Restriction Endonucleases 4.1.2 Sticky and blunt ends Figure 4.2 Reading a palindromic sequence Almost all restriction enzymes are considered palindromic , reading the same forwards and backwards like verbal palindrome “radar” In the strict sense, restriction enzymes are not palindromes because they read the same from both the top and bottom strands (eg. EcoRI enzyme reads “GAATTC” from the 5’ to 3’ direction) taking in account that the two strands of DNA run in opposite directions
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4.1.2 Sticky and blunt ends Figure 4.3 Sticky ends generated by EcoRI EcoRI cuts between G and A on each strands, leaving 4 base 5’ overhang of 5’AATT3’ on both strands, which are complementary to each other. These complementary ends are
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Chapter_4___Cutting_and_joining_DNA_v2 -...

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