Recombinant DNA technology

1. Recombinant DNA technology • DNA has now become the easiest of the cellular macromolecules to be analyzed • possible to cut out a region of DNA containing a specific gene, to produce virtually unlimited number of exact copies, and to determine its nucleotide sequence; to alter or redesign the gene and transfer it back into cells in culture or insert it into animals and plants. • Provides a means to study the function of proteins and their domains • Used to detect mutations that are responsible for genetic diseases, in forensic science to identify or acquit suspects, to produce pharmaceuticals like insulin for diabetics.

2. Restriction nucleases catalyze the hydrolysis of a phosphodiester bond in a nucleic acid at particular site, determined by a short sequence (often palindromic). Most were isolated from bacteria and are named for there source.

3. A given restriction nuclease will always cut a given DNA molecule at the same sites resulting in the same set of DNA fragments. • The sequence at which each restriction nuclease will cut is short, usually 4-8 base pairs. • Hae III cuts at a sequence of 4 nucleotide pairs. This would be expected to occur purely by chance about 1 out of 256 nucleotide pairs ( 1 in 44) probability is 1 in 4n

4. Agarose or polyacrylamide gels contain a microscopic network of pores. Voltage is applied and negative DNA travels toward the positive electrode. Gel electrophoresis separates DNA fragments of different sizes. Visualized by soaking gel in a dye that binds DNA and fluoresces under ultraviolet light. Visualized by labeling DNA with radioactive phosphorus atoms prior to isolation, ex. replicating virus in the presence of 32P - autoradiography Larger fragments move more slowly, held back by the gel’s matrix

5. Construction of physical maps of DNA includes charting the position of various landmarks present along it, including known genes and restriction sites. Physical maps created by treating DNA with different combinations of restriction nucleases are called restriction maps. This is a restriction map of the two alpha globin gene (red) cluster. Each restriction enzyme used is designated by a letter.

6. Enzymatic method of sequencing DNA excess many reactions result in many fragments ending in A, each a different size depending on where the ddATP happens to encorporate

7. Determining the complete sequence of a DNA fragment: denatured to ssDNA added

8. Complete sequences of several bacterial genomes, Drosophila (fruit fly), and C elegans (worm) have been achieved in this way. Human genome is being sequenced. This is a representation of the complete nucleotide sequence of the genome of S. cerevisiae.

9. Denaturation and renaturation (hybridization) of DNA Allows detection of specific sequences in DNA or RNA

10. DNA hybridiqation facilitates diagnosis of genetic diseases including prenatal diagnosis. This requires a probe, a short single-stranded DNA (oligonucleotide, 10-1000 nucleotides long). These (up to 120 nucleotides) can now be synthesized using a machine. If probes are short (20 nucleotides) they can be hybridized at temperatures at which only perfectly matched DNA be stable. Who is a carrier, who is effected? southern blot

11. Detection of specific DNA fragments by gel-transfer hybridization (southern blotting). Alkali solution is sucked up through the gel and nitrocellulose paper, into the paper towels. It denatures the DNA and transfers the single stranded fragments from the gel to the nitrocellulose sheet where it firmly adheres.

12. Chromosomes are briefly exposed to very high pH to separate the two strands. Nucleotide probes have been chemically labeled and are allowed to bind to the chromosomes. Fluorescent antibodies that bind the chemical on the probe are used to visualize the location of that particular sequence. In situ hybridization to locate genes on chromosomes. These are metaphase chromosomes.

13. In situ hybridization can also reveal the distribution of a particular mRNA in cells in tissues. This is a group of cells in a growing shoot tip of a snapdragon. Only a few cells express the mRNA for cyclin, a protein that triggers cells to divide. The probe has been linked to an enzyme that produces a dark blue reaction product when a substrate is added.

14. formed by Eco RI formed by Hae III and Alu I Formation of recombinant DNA molecules in vitro

15. Bacterial plasmids can be used to clone DNA (make identical copies of).

16. fragment to be cloned is cut by the same restriction nuclease. can this fragment go in the plasmid “forward” and “backward”?

17. Cloning of a DNA fragment

18. How do we clone a human gene (ex. – factor VIII, mutated in hemophilia A).? 1. break up the total genomic DNA into smaller more manageable pieces – create a DNA library. Since this is the entire human genome is is called a genomic library. Many genomic libraries for many different organisms exist and are shared between researchers.

19. at a concentration which leads to no more than one plasmid per bacteria. Why is this important?

20. How do we find the gene for Factor VIII? We don’t know the sequence. One thing to do is purify some protein from human blood donors. A small portion of this protein was then sequenced. The genetic code was applied in reverse. Probes are prepared by chemical synthesis.

21. Go back to the living colonies and pick up the ones positive for the the probe.

22. This may lead to the cloning of only part of the gene, as happened for Factor VIII. • Often more work has to be done to piece together the whole gene, introns and exons. • Can you think of a way to take the DNA sequence you have and “go fishing” for all the exons that encode Factor VIII without the introns in the way? • A gene can be large due to introns, it may be advantagous to have the sequence of the exons only. Where in the cell will you find this?

23. It is relatively easy to create a cDNA library. Since Factor VIII is made in the liver, cDNA from liver tissue was used to isolate the mRNA sequence by using the DNA fragment we cloned in the last figure.

24. Several important differences between a genomic library and cDNA libraries. • 1. genomic = same sequences regardless of cell type cDNA = only sequences that are expressed • 2. genomic = contains large amount of repetitive DNA, introns, gene regulatory regions, spacer DNA in addition to exons. cDNA = contain only coding sequences and only those for genes that are transcribed in this particular tissue or cell type, even giving an indication of the level of transcription • 3. Only cDNA contains uninterrupted coding sequence of the gene. Can be used directly for expression in bacterial or yeast cells which can not remove mammalian introns. Can be used directly to deduce the amino acid sequence.

25. genomic cDNA

26. Different hybridization conditions allow less than perfect DNA matching. New genes may have arisen during evolution by duplication and divergence of existing genes and by using gene segments (domains) from one gene in new combinations (as discussed in the last chapter). Most genes have a family of related members often with related function. Finding these genes, or a gene from one species in another, can be done by differential hybridization using sequences from the known gene as DNA hybridization probes for the unknown genes.

27. Polymerase chain reaction (PCR) is now quicker and less expensive thatn the cloning methods we have already discussed. However, you must have the beginning and ending sequences in most cases. cooling in presence of large excess of two DNA primers DNA polymerase used is isolated from a thermophilic bacterium. It is stable at higher temperatures than eucaryotic DNA polymerases. It does not need to be added at each cycle, it remains active through the heating cycles.

28. Extra Each cycle doubles the amount of DNA synthesized. A single cycle takes about 5 minutes and the whole process is now automated. PCR is very sensitive, it can detect a single copy of a DNA sequence in a sample.

29. PCR can be used to obtain a genomic or cDNA clone. Because of its sensitivity, this can be used to determine what mRNA a cell is expressing, sometimes an indication of protein expression. This technique is not quantitative.

30. PCR can be used to detect the presence of a viral genome in a sample of blood.

31. PCR can be used in forensic science. DNA from a very small sample of blood, skin, saliva, hair, or semen. The DNA sequences that create the variability used here contain short, repeated sequences such as GTGTGT..., which are found in various positions in the human genome (VNTR). The number of repeats in each run is highly variable in the population. The length of each fragment depends on the number of repeats in this area. variable number of tandem repeat

32. DNA bands are obtained from a set of different PCR reactions, each that amplifies DNA from a different VNTR locus. As you do more separate VNTRs on each sample the odds of coming up with a match become virtually impossible.

33. DNA engineering: New DNA can be made from the combination of naturally occurring DNA sequences or chemically synthesized DNA. cloned to purify and amplify the new DNA cloned to purify and amplify the new DNA cloned to purify and amplify the new DNA

34. Rare cellular proteins can be made in large amounts for study and for clinical. An expression vector is used. These cloning vectors include appropriate gene regulatory and promoter DNA sequences necessary to enable an adjacent protein-coding DNA insert to be efficiently transcribed in cells. This can then be translated in the cell. Different cells require different regulatory sequences. Some proteins require modification, and therefore must be expressed in eucaryotic cells. The vector can be inserted into the cell (transient expression) or actually incorporated into the genome. Bacterial, yeast, insect, or mammalian cells are used. The protein can be easily purified after lysis of the cells or it can be secreted.

35. Often, small amounts of a protein is isolated. In order to study it, a short partial amino acid sequence is obtained. This is used as a probe to screen a genomic or cDNA library. Once the gene or cDNA is cloned, it can be inserted into an expression vector and introduced into a cell line for large scale production.

36. What does a protein do? One way to look at this question is to mutate the protein. Mutants can be made temperature-sensitive, so that the mutation only shows when the temperature is increased (or decreased). Now, instead of starting with a random mutation, we can start with a cloned gene and make mutations in vitro. We can introduce precise mutations, change one specific amino acid. This is site-directed mutagenesis. This can be used in single cells and by creating organisms which express this particular mutation.

37. The “ultimate test” of the function of a gene mutated is to insert it into the genome of an organism (transgenic) and see what happens. NOT ULTIMATE! What problems do you see?

38. Transgenic techniques make it possible to produce complex organisms that are missing certain gene products entirely, express the protein only in certain cells, or express a mutated form of the protein. embryonic stem cells homologous recombination

41. Transgenic animals often are not viable due to the importance of the knocked out protein for development. Here, a gene knocked out of both chromosomes leaves the developing mouse with no cerebellum. This is a common problem when you are looking for the function of a protein in an adult mouse – redundancy.