Fun with DNA polymerase

Transcription

1 Fun with DNA polymerase Why would we want to be able to make copies of DNA? Can you think of a situation where you have only a small amount and would like more? Enzymatic DNA synthesis To use DNA polymerase in vitro, one needs a starting point for the replication; then use the enzyme to extend it. The primer is simply a short (about 20 nucleotides) piece of DNA that is complementary to a specific template DNA location. Using available free nucleotides, the DNA polymerase makes a copy of the template DNA starting at the primer location, just as it does during DNA replication! the DNA copy can be labeled by adding labeled free nucleotides to the mixture 1

2 PCR recipe Enzymatic DNA synthesis of DNA can be repeated many times using the polymerase chain reaction. Start with a mixture of: template dsdna primers (probes for the template ends) free nucleotides (A, T, G, and C) DNA polymerase Illustration of PCR (1) Heat to 95 C, dsdna comes apart to form ssdna templates. (2) Complementary DNA primers attach to templates when you cool to about 60 C. (3) DNA polymerase will make copies of DNA starting at the location of an attached primer. (4) Repeat steps 1-3. The number of copies approximately doubles after each cycle. Only DNA that is complementary to the added primers is duplicated. 2

3 Taq DNA polymerase Enzymatic DNA synthesis has been possible in vitro for decades but was not economical because heating the dsdna to 95 C caused the DNA polymerase to denature (unfold) and it would not re-fold correctly after it was cooled down. This meant that fresh DNA polymerase would have to be added for each cycle. The solution was using DNA polymerase from a bacterium that lives in in hot hot springs (Thermus aquaticus). It It had evolved enzymes that remained functional at at high temperatures. This clever approach by by Kary Mullis was published in in 1988 and won the the Nobel Prize in in It It has has revolutionized biotechnology and molecular medicine. Tools of Genetic Engineering Cutting, Pasting, and Inserting DNA 3

4 Guiding Concepts (1) DNA interactions are simple: complementary sequences attach, or hybridize. TCGTGCCTT small DNA probe + ---GCCTTAGCACGGAATTGAACCGGC--- ssdna TCGTGCCTT ---GCCTTAGCACGGAATTGAACCGGC--- (2) Use naturally-occurring enzymes to cut, copy, and join DNA. e.g. DNA polymerase universal genetic code allows us to mix & match enzymes and genetic material from different organisms, even in vitro. Illustration of DNA cloning procedure Cloning vector: DNA from source organism is cleaved with a restriction endonuclease and inserted into a plasmid DNA construct. Transformation: The resulting DNA construct is introduced into a target host cell. Selection: The cells that carry the construct are identified and grown. The protein is produced and harvested. 4

5 The role of cutting up DNA is performed by enzymes called restriction endonucleases. For what purpose do you think these enzymes evolved in wild bacteria? GREEN: making small DNA fragments that can be used by ribosomes instead of RNA. RED: chopping up viral DNA as a defense against infection. YELLOW: cutting chromosomal DNA into small pieces that can be replicated more easily during cell division. Cutting DNA with restriction enzymes Restriction endonucleases cleave dsdna at specific, recognized locations, generally palindromic sequences. ( A man, a plan, a canal, Panama. ) The enzymes were discovered in E. coli bacteria in the 1970 s. Their role in bacteria is defense against viral invasion. Viruses inject their own DNA or RNA into a cell, the restriction enzymes can chop them up. Hundreds of different restriction enzymes are now known, with a wide variety of recognition sequences. This gives molecular engineers a varied selection of precision DNA cutting tools. 5

6 Illustration of endonuclease activity The EcoRI restriction enzyme recognizes the following sequence: -G-A-A-T-T-C- -C-T-T-A-A-G- The complementary strand is simply the reverse sequence, thus palindromic. DNA fingerprinting Used to characterize biological samples for legal proceedings; to match sample DNA with DNA taken from a defendant or to rule out the possibility of a match. Also used for paternity testing since the DNA fingerprint of a child is a composite of the patterns of the mother and father. In principle, the entire chromosomal DNA (genome) could be sequenced and compared, but that would be outrageously expensive. Also only recently possible. 6

7 To make a DNA fingerprint (1)Digest sample DNA with a restriction endonuclease. This results in a distribution of fragments with various sizes. (2)These fragments are separated by size by placing them on a gel exposed to an electric field that forces them to move. Smaller fragments move through the gel more easily and travel farther. (3)Labeled probes are added that bind to certain sequences within the fragments. (4)Images of the radioactive labels show distinctive bands that indicate the sizes of the fragments that represent the distinctive distances between restriction enzyme binding sites. The resulting image is like a histogram of fragment sizes. DNA fingerprint bands large fragments small fragments 7

8 Joining DNA fragments Sticky ends formed by restriction enzymes help hold fragments in place temporarily, but the DNA ligase enzyme is necessary to truly glue the new dsdna together. -CTATG -GATACTTAA hybridization AATTCTTGAC- GAACTG- nick -CTATGAATTCTTGAC- -GATACTTAAGAACTGnick DNA ligase (ligation) -CTATGAATTCTTGAC- -GATACTTAAGAACTG- So far we can make the DNA cloning vector How do we get it inside the cell? 8