RDNA Week 9 L2 PDF

RDNA week 9 L2 https://www.iflscience.com/genetically-modified-purple-tomatoes-will-be-popping-up-on-us-plates-by-next-spring-65283  Six steps of Recombinant DNA 1. Isolating (vector and target gene) 2. Cutting (Cleavage) 3. Joining (Ligation) 4. Transforming 5....

RDNA week 9 L2 https://www.iflscience.com/genetically-modified-purple-tomatoes-will-be-popping-up-on-us-plates-by-next-spring-65283  Six steps of Recombinant DNA 1. Isolating (vector and target gene) 2. Cutting (Cleavage) 3. Joining (Ligation) 4. Transforming 5. Cloning 6. Selecting (Screening) Use of Restriction Enzymes & DNA Ligase to make recombinant DNA molecules Purified and characterized the cleavage site of a Restriction Enzyme.  What are restriction enzymes? Molecular scissors that cut double stranded DNA molecules at specific points.  Restriction Enzymes Restriction endonucleases cuts DNA at specific nucleotide sequences = restriction sites. REs are found in many different strains of bacteria where their biological role is to participate in cell defence. Enzymes selectively cut up (restrict) foreign (e.g. viral) DNA that enters the cell, in a process called restriction. prevent the replication of the phage by cleaving its DNA at specific sites. Host DNA is protected by Methylases which add methyl groups to A or C bases modifying the site and protecting the DNAfrom cleavage. To cut the DNA, restriction enzyme makes two incisions, each strand of the DNA double helix. Biological role of REs  Restriction Enzymes scan the DNA sequence Find a very specific set of nucleotides Make a specific cut  Picking a palindrome Words that read the same forwards as backwards Hannah hannaH Level leveL Madam madaM  Palindromes in DNA sequences Genetic palindromes are similar to verbal palindromes. 5’ 3’ A palindromic sequence in DNA is one in which the 5’ to 3’ base pair sequence is identical on both strands. Restriction enzymes recognize and make a cut within specific palindromic sequences, known 3’ 5’ as restriction sites, in the DNA. This is usually a 4- or 6 base pair sequence.  Cleavage Patterns of Some Common Restriction Endonucleases Restriction fragments can be blunt ended or sticky ended © 2012 Pearson Education, Inc. Figure 20.1  Cohesive (‘sticky’) ends These are products of restriction enzyme digestion with protruding ends these ends are known as cohesive, or ‘sticky’ ends since they can bind to any other end with the same overhanging sequence, by base pairing (annealing) of the single-stranded tails For example, any fragment formed by an EcoRI cut can anneal to any other fragment formed in the same way, and may subsequently be joined covalently by ligation In some cases, DNA ends formed by enzymes with different recognition sequences may be compatible, provided the single-stranded tails can base-pair together.