1. Manipulating the Genome: DNA Cloning and Analysis 20.1 – 20.3 Lesson 4.8

2. Manipulating the Genome Recombinant DNA – DNA in which nucleotide sequences from two different sources are combined in vitro into the same DNA molecule Genetic Engineering – direct manipulation of genes for practical purposes Biotechnology – manipulation of organisms or their components to make useful products – Pest resistance in crops, bacteria to clear toxic waste, protein that dissolves blood clots in the heart, human growth hormone for stunted growth

3. You Too Can Clone! A plasmid is isolated from a bacterial cell and the foreign DNA (that you want to clone) is inserted into it. This plasmid replicates, carrying the desired gene and it is duplicated

4. Restriction Enzymes Enzymes that cut DNA molecules at a limited number of specific locations – Each one recognizes a restriction site – Cuts them into restriction fragments that always cut in the same place – Resulting double-stranded restriction fragments have at least one single-stranded sticky end Can form complimentary sticky ends on any other DNA molecules, made permanent by DNA ligase

6. Detailed Steps in Cloning Cloning Vector – original plasmid that can carry foreign DNA into a cell and replicate there. In the following example: a human gene is inserted into a plasmid from E. coli. The plasmid contains amp R gene, which makes E.coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes B- galactose. This enzyme hydrolyzes a molecular mimic of lactose to form a blue product. Only 3 plasmids and 3 human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples.

8. How to Identify Clones Carrying the Gene of Interest Nucleic Acid Hybridization – DNA of the gene of interested is detected by its ability to base- pair with a complimentary sequence on a nucleic acid probe – Scientists will synthesize a probe complimentary to the gene of interest

9. Genomic Library Previous approach to cloning is called “shotgun” approach, because so many recombinant plasmids exist (white colonies on the dish), while waiting for the target plasmid to grow (blue). Genomic Library – the complete set of plasmid clones, each carrying copies of a particular segment from the initial genome

10. Another Form of Cloning Extracts mRNA from cells, uses reverse transcriptase (from retroviruses) to form a DNA transcript from the mRNA, then DNA polymerase to form a complementary strand of DNA. This double stranded DNA made from mRNA is called complemetary DNA (cDNA) This is then inserted into vector DNA like the previous method

11. Cloning vs. Amplification Cloning works well to prepare large quantities of a particular gene, but if the DNA source is not pure or there isn’t much of it, PCR (polymerase chain reaction) is used. Joey’s video at 1:15

12. Restriction Fragment Analysis Once we have large amounts of DNA, we could start to see what those genes code for, where they are located, and the evolutionary history of the gene, but to do that, the nucleotide sequences must be determined. Gel Electrophoresis – uses gel as a molecular sieve to separate nucleic acids or proteins on the basis of their size, electrical charge and other physical properties

14. How is Gel Electrophoresis useful? Ex: Sickle Cell Anemia is a mutation of a single nucleotide located in a restriction sequence of the B- globin gene, causing the restriction enzymes to break up the DNA in different sizes.

15. Southern Blotting If you want to compare more DNA (like between 3 people), gel electrophoresis doesn’t give a clear picture, so southern blotting is used - combines gel electrophoresis and nucleic acid hybridization to make the bands more separately clear

17. RFLPs Restriction Fragment Length Polymorphisms - differences in the restriction sites on homologous chromosomes that result in different restriction fragment patterns – Located in non-coding DNA – Inherited markers that identify certain populations or heredity (paternity tests) – Used these to begin mapping the human genome – Now have, human, E. coli, yeast, nematode, fruit fly, mouse

18. Genomics Study of whole sets of genes and their interactions – Possible now that the entire genome is sequenced

19. Curiosity of Genome Size Genome size usually increases from prokaryotes to eukaryotes, but genome size does not always correlate with biological complexity 120 x 10 9 base pairs 40 times less than Fritillaria assyriaca

20. Curiosity of Genome Size 11,000 genes in genome25,000 in genome

21. Genome Size & Estimated Number of Genes ORGANISMGENOME SIZE (Mb)NUMBER OF GENESGENES PER Mb Bacterium1.81700940 E. coli4.64400950 Yeast125800480 Nematode9719,000200 Arabidopsis thaliana (plant) 11825,500215 Fruit fly18013,70076 Rice43060,000140 Zebrafish170022,00013 House mouse260025,00011 Human290025,00010 Fritillaria assyriaca120,000ND

22. Why are humans so much more complex? Gene expression is much more regulated in humans Splicing capabilities in humans causes great diversity Larger capability for interaction of gene products

23. Determining Gene Function Usually, the gene is disabled, and the cell is observed to see what consequences result – Can be done through in vitro mutagenesis (specifically adding mutations to stop that gene from coding) – Can be done through RNA interference (RNAi) – blocks the mRNA from forming in transcription, so the gene’s product can’t be produced

24. Determining Which Genes Are Expressed.