2 Plasmid Use Plasmids are good tools for DNA Technology Can be isolated from bacterial cellsOften carry resistance genesIsolated genes of interest can be inserted into the plasmidHow is this insertion done?Restriction endonucleases (enzymes)
3 Restriction Enzymes Where were restriction enzymes first found? Bacterial cellsThey were used to protect bacteria from intruding phage DNABacterial DNA is modified to protect it from its own restriction enzymesRestriction enzymes often cut DNA leaving “sticky ends”
4 Restriction sites are the regions on the DNA that the restriction enzyme cuts. Why are restriction sites palindromes?
5 How are restriction enzymes used in DNA technology?
14 Gel electrophoresis How does gel electrophoresis work? Uses electric charge to separate molecules based on their sizeWhat charge does DNA have?NegativeWhich sized fragments will move furthest through the gel?Smallest ones
16 Restriction Fragment Analysis Genetic markers are regions of DNA that vary from person to personUsually located on non-coding regions of the DNAUsing restriction enzymes and gel electrophoresis, DNA of different individuals can be analyzed and comparedExtract DNA and treat it with restriction enzymes
17 The red triangles indicate where the enzyme cuts the DNA. Procedure: The restriction enzyme is added to the DNA being analyzed and incubated for several hours, allowing the restriction enzyme to cut at its recognition sites. The DNA is then run through a gel, which separates the DNA fragments according to size. You can then visualize the size of the DNA fragments and assess whether or not the DNA was cut by the enzyme.Gel with an uncut and cut samples of DNA. Note that the sizes of the cut DNA fragments add up to the size of the uncut DNA.
18 How could you detect the differences between these 2 alleles?
20 Using RFLP Analysis to Detect Harmful Alleles Harmful, disease causing alleles usually have identical RFLP’s within a familyOnce the known RFLP’s for the normal and disease causing alleles are known family members can be tested using Southern Blot analysis
25 A Radioactively labeled probe is added to the nylon membrane The probe is either RNA or DNA that will compliment a specific bp sequence on the DNAAfter unbound probes have been washed away only bound probes remain on the blot
26 VNTR A VNTR is variable numbered tandem repeat Tandem repeats are interspersed throughout the genomeDifferent VNTR’s can be detected using southern blot technique
28 One VNTR is inherited from each parent Southern blot analysis usually shows 2 different bands one inherited from each parentHow could an individual have one band for the VNTR?He/She inherited the same sized VNTR from each parent
29 Three different alleles for this particular VNTR What are the different genotypes for these 6 individuals?
31 Frequency of VNTR’sFrequency of allele pattern at a single VNTR has been established for specific sites within the genomeWhat is the probability of matching a 5 locus DNA profile, where each locus is:0.01, 0.02, 0.03, and 0.10?One in 27.8 million people will randomly match this profileOJ’s profile was of 24 different loci and he matched all 24!The odds were 1 in 10 billion
32 Amplification of DNAWhat enzymes would be necessary for DNA amplification?DNA polymerase and ligaseWhat else would be necessary for the process to work?Primers and nucleotides!How was the original DNA initially uncoiled and unwound?HeatWhy did the heat cause difficulty with the procedure?
47 Supplemental Lab 6AIn this lab we will transform E. coli bacterial colonies with recombinant plasmid DNAIt will be your job to distinguish between bacterial colonies that have been transformed with the recombinant plasmids