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Lab Techniques James Chappell & Cheuk Ka Tong. Contents Page 1.Restriction Enzymes 2.Gel Electrophoresis 3.Blotting techniques-Southern, Northern and

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Lab Techniques James Chappell & Cheuk Ka Tong Slide 2 Contents Page 1.Restriction Enzymes 2.Gel Electrophoresis 3.Blotting techniques-Southern, Northern and Western 4.DNA sequencing 5.Polymerase Chain Reaction (PCR) 6.Recombinant DNA 7.Gene Cloning 8.References/ Recommended Reading Slide 3 Restriction Enzymes Restriction Nuclease - An enzyme that cleaves a molecule of DNA at any site where a specific short sequence of nucleotides occurs. 2key types Endonuclease- Cleaves within the DNA molecule Exonuclease- Cleaves at the ends of the DNA molecule Slide 4 Endonucleases 4 types - classified on subunit composition, cleavage position, sequence-specify and co-factor requirement. Type II is the main one that is used in gene cloning. Two key terms Recognition sites Nucleotide sequence that is recognised. Cleavage sites- Phosphodiester bond that is cleaved. Slide 5 Endonucleases Make break the phosphodiester bond of each of the stands of the double helix. 5----GAATTC----3 3----CTTAAG----5 5----G-3 5AATTC----3 3----CTTAA--5 3G----5 Slide 6 Endonucleases- Recognition site + Cleavage site Slide 7 Endonucleases- Recognition site 3 Considerations- Sequence Determine specificity Length of sequence- Determines frequency Palindrome- sequence that reads the same backwards and forwards. Isoshizomers Restriction enzymes that recognise the same recognition site Slide 8 Endonucleases- Cleavage site Slide 9 Endonucleases Applications Allow specific cutting and removal of genes from a complex molecule of DNA. Complementary sticky ends (cohesive ends) allow joining of DNA molecules. "The work on restriction nucleases not only permits us easily to construct recombinant DNA Molecules and to analyze individual genes but also has led us into the new era of synthetic biology where not only existing genes are described and analyzed but also new gene arrangements can be constructed and evaluated" Nobel prizes and restriction enzymes in GENE (1971) Slide 10 Gel Electrophoresis Electrophoresis - the migration of charged molecules in an electric field though a solution or solid support Various types defined by support used 1.Paper amino acids, small peptides 2.Polyacrylamide Proteins, small DNA/RNA ( Polymerase Chain Reaction (PCR) Five noteworthy features of PCR: 1)The sequence of the target need not be known. 2)The target can be much larger than the primers (>10 kb). 3)Primers do not have to perfectly match flanking sequences. 4)Stringency can be controlled by temperature and salt (MgCl 2 ). 5)PCR is very sensitive. Slide 26 Recombinant DNA Technology The construction of new combinations of unrelated genes. These novel combinations can be cloned and amplified by introducing them into host cells. Slide 27 Recombinant DNA Technology A DNA fragment of interest is covalently joined to a DNA vector. - A vector can replicate autonomously in an appropriate host. - Plasmids and phage are common vectors for cloning in E.coli. Slide 28 Recombinant DNA Technology The DNA fragment of interest and the plasmid vector are both cut using the same restriction enzyme. The single-stranded ends of the fragment are complementary to those of the cut plasmid. The DNA fragment and the cut plasmid are annealed and then joined by DNA ligase. Slide 29 Gene Cloning One of the most useful plasmids for cloning is pBR322. pBR322 contains genes for resistance to tetracycline and ampicillin. Different endonucleases can cleave this plasmid at a variety of unique sites. Slide 30 Gene Cloning Insertion of DNA at the EcoRI site does not alter either of the genes for antibiotic resistance. However insertion at the SalI or PstI site causes insertional inactivation. Basis for selection of cells containing recombinant DNA. Slide 31 References/ Recommended Readings 1.Chapter 8 Manipulating Proteins, DNA and RNA - Molecular Biology of the Cell (4 th Edition), Alberts, Johnson, Lewis, Raff, Roberts, Walter, Garland Science 2.Chapter 6- Exploring Genes -Biochemistry (5 th Edition), Berg, Tymoczko, Stryer, Freeman

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