TOOLS OF BIOTECHNOLOGY HLeeYu Jsuico Junsay Department of Chemistry School of Science and Engineering Ateneo de Manila University 1
Biotechnology is the applicaaon of biological macer for useful operaaons. 2
Biotechnology is the applicaaon of biological macer for useful operaaons. 1. How do you sequence DNA? 2. How do you visualize DNA? 3. How do you make more copies of DNA? 4. With all these in mind, can you change an organism s genes? 3
DNA SEQUENCING AND VISUALIZATION 4
DNA can be characterized by size by means of electrophoresis. SIZE MARKERS + + + + + + + + + + + + 5
Agarose Gel Electrophoresis (AGE) is the most common technique for DNA separaaon and size determinaaon. 6
DNA is visualized by means of UV acave agents that bind to DNA. This can be viewed under UV light. 7
DNA sequences can be probed for specific sequences thru blo_ng methods (Southern BloCng by Edwin Southern) 8
DNA sequences can be probed for specific sequences thru blo_ng methods (Southern BloCng) Probe A labeled single strand of DNA or RNA seek out a complementary sequence of single stranded DNA Used in screening Primer Single stranded Required by DNA Polymerase to synthesize DNA 9
DNA sequences can be probed for specific sequences thru blo_ng methods (Southern BloCng) NORTHERN BLOT = RNA WESTERN BLOT = PROTEIN EASTERN BLOT =?!? SOUTHERN BLOT = DNA 10
The Sanger method makes use of a DNA you want to sequence, a short primer, DNA Polymerase, dntps and specific ddntps. 11
The Sanger method makes use of a DNA you want to sequence, a short primer, DNA Polymerase, dntps and specific ddntps. 12
The Sanger method makes use of a DNA you want to sequence, a short primer, DNA Polymerase, dntps and specific ddntps. 13
The Sanger method makes use of a DNA you want to sequence, a short primer, DNA Polymerase, dntps and specific ddntps. 14
DNA AMPLIFICATION: POLYMERASE CHAIN REACTION 15
A rapid and versaale in vitro method to amplify defined target DNA within a heterogeneous collecaon of DNA sequences (genomic DNA or cdna) was invented by Kary Mullis in 1984. He called it polymerase chain reacson 16
5 3 DNA fragment (blue) with sequence of interest (orange) sense 3 anasense 5 5 3 Taq Polymerase 3 5 Primers Free dntps Figure 1. Dissolve all your components in a buffer soluaon and place them in PCR Tubes. Load PCR tubes in PCR machine hcp://www.molecularstaaon.com/molecular biology images/data//509/pcr tubes1.png hcp://www.molecularstaaon.com/molecular biology images/data/509/pcr machine.jpg
5 sense 3 3 anasense 5 Figure 2. PCR cycles through 3 temperature steps. The first of which is the Denaturing Step: Hold at 94C for 1 minute to separate the double stranded DNA to its individual strands. 5 3 5 sense 3 3 5 3 anasense 5 Figure 3. The second step is the Annealing step: Lowering temperature to 50 65 C depending on the melang temperature of the primers, holding for about 1 minute. This step allows for the acachment of the primers to sequence of interest.
5 3 5 sense 3 3 5 3 anasense 5 5 5 sense 3 3 anasense 5 Figure 4. The third step is the Extending step: increase temperature to around 75 80 C (or wherever the enzyme operates at its opamum). Hold for a variable Ame depending on the length of sequence to be amplified. It usually takes a polymerase 1000 bp per minute. At this step, free dntps are used by the polymerase to make copies of the DNA of interest. 5
Figure 5. This shows the temperature profile of the PCR process with the corresponding steps involved. hcp://www.mun.ca/biology/scarr/pcr_sketch_3.gif
5 3 sense copy 5 5 sense 3 anasense copy 3 3 anasense 5 Amer cycle 2 5 sense 3 3 5 anasense copy 2 sense copy 2 5 3 3 5 anasense copy sense copy 5 3 3 5 anasense copy 2 sense copy 2 5 3 3 anasense Figure 6. Amer 1 cycle, you have now two copies of your DNA. The first cycle creates a copy with extraneous sequence (in dark green). Another cycle will create a fragment that only contains your sequence of interest (orange), and this will be further amplified to create fragments of your desired length. 5
Figure 7. This an agarose gel of 3 Assue samples ran in PCR with two primer sets. NoAce that only Tissue 2 and Tissue 3 contain fragment of interest as compared to a posiave control. One can also see, heavier fragments (encircled in red) which could be those longer fragments with extraneous DNA. Primer set 1 Primer set 1 Primer set 2 Primer set 2 DNA Molecular Weight Ladder hcp://upload.wikimedia.org/wikipedia/en/d/d0/roland_gel.jpg
hcp://users.ugent.be/~avierstr/principles/pcrcopies.gif Figure 8. Number of fragments exponenaally grow unal a plateau due to limitaaons in the amount of components (dntps) hcp://www.ionchannels.org/content/images/14 02.jpg
RECOMBINANT DNA TECHNOLOGY 24
The ability to change the geneac characterisacs of fundamental life forms is generally called recombinant DNA technology or genesc engineering. 25
Making recombinant DNA consist of the covalent inseraon of a DNA fragment from one type of cell or organism into the replicaang DNA of another type of cell. 26
Making recombinant DNA consist of the covalent inseraon of a DNA fragment from one type of cell or organism into the replicaang DNA of another type of cell. The carrier of the geneac material is called a vector. RestricSon enzymes are endonucleases which cut the DNA at specific palindromic sites. 27
Making recombinant DNA consist of the covalent inseraon of a DNA fragment from one type of cell or organism into the replicaang DNA of another type of cell. 28
Two common vectors are in common use: Plasmids and Bacteriophage Plasmids (1) should be able to replicate in a relaxed fashion, (2) should be small, (3) should contain markers for screening progeny and (4) should only have one cleavage site. 29
Two common vectors are in common use: Plasmids and Bacteriophage Bacteriophage or λ phage is usually larger, can make many copies of the recombinant DNA and have efficient packaging 30
RestricAon enzymes cut at specific sites and produces blunt or sacky ends. 31
RestricAon enzymes cut at specific sites and produces blunt or sacky ends. 32
Blunt ends are hard to ligate to DNA, homopolymer tails may used instead. 33
Recombinant DNA must be introduced to a cell by means of several methods, then these cells must be grown and selected. 34
Recombinant DNA must be introduced to a cell by means of several methods, then these cells must be grown and selected. 35
Recombinant DNA must be introduced to a cell by means of several methods, then these cells must be grown and selected. 36
Recombinant DNA must be introduced to a cell by means of several methods, then these cells must be grown and selected. 37
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Figure 8-39 Molecular Biology of the Cell ( Garland Science 2008)
Figure 8-40 Molecular Biology of the Cell ( Garland Science 2008)
Figure 8-41 Molecular Biology of the Cell ( Garland Science 2008)