Molecular Cloning. Genomic DNA Library: Contains DNA fragments that represent an entire genome. cdna Library:

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1 Molecular Cloning Genomic DNA Library: Contains DNA fragments that represent an entire genome. cdna Library: Made from mrna, and represents only protein-coding genes expressed by a cell at a given time.

2 Molecular Cloning BIO Lecture 09/26/2006

3 Molecular Cloning Expression Library Made with a cloning vector that contains the required regulatory elements for gene expression, such as the promoter region. Promoter cdna genes Can insert into host cells to produce a protein or create a library. Useful for identifying a clone containing the gene or cdna of interest.

4 Molecular Cloning Use of Reporter (marker) gene: Identify regulatory elements: The regulatory sequence of a cloned eukaryotic gene is ligated to a reporter gene that encodes an easily detectable enzyme. The resulting plasmid is then introduced into cultured recipient cells by transfection. An active regulatory sequence directs transcription of the reporter gene, expression of which is then detected in the transfected cells.

5 Molecular Cloning Use of Reporter (marker) gene: β-galactosidase gene - Break down X-gal to produce blue color. Luciferase gene found in firefly and the bacteria, the protein produces light in response to the molecules luciferin and ATP. Green fluorescent protein (GFP) produced by the jellyfish and interacts with the protein aequorin to produce fluorescence. GFP β-glucuronidase gene (GUS) encodes an enzyme that breaks down chemicals called β-d-glucuronides. Can produce a blue or fluorescent color.

6 Molecular Cloning Use of Reporter gene: Identify regulatory elements:

7 Molecular Cloning Use of Reporter gene: Identify trans-acting factors: Searching for gene (X-protein) that can trans-activate the expression of another gene under control of X-binding site. Plasmid 2 : Encode a reporter gene under a control of X-binding site. Plasmid 1 : Expression library. Expression of reporter gene from plasmid 2 can be achieved when plasmid 1 encode for X factor which interact with X binding site. Same strategy can be used to identify a repressor protein.

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10 Molecular Cloning BIO Lecture 09/26/2006

11 Molecular Cloning Random primed (internal) labeling : The Klenow subunit of E. coli DNA polymerase I can synthesize new radiolabeled DNA strands using as a template separated strands of DNA, and random hexanucleotide primers.

12 Molecular Cloning Run-off transcription from cloned DNA inserts: Labeled insert-specific RNA transcripts can be generated using SP6 RNA polymerase and a cocktail of NTPs, at least one of which is labeled (UTP in this case).

13 Molecular Cloning End labeling: (A) Kinase end-labeling of oligonucleotides. The 5 terminal phosphate of the oligonucleotide is replaced in an exchange reaction by the 32Plabeled γ-phosphate of [γ- 32P]ATP. The same procedure can be used to label the two 5 termini of double-stranded DNA. (B) Fill-in end-labeling by Klenow. The DNA of interest is cleaved with a suitable restriction nuclease to generate 5 overhangs. The overhangs act as a primer for Klenow DNA polymerase to incorporate labeled nucleotides complementary to the overhang.

14 DNA Sequencing Developed by two groups in 1977: 1. Allan Maxam and Walter Gilbert developed a chemical method that modifies and removes a specific base from the DNA strand. The pieces that are generated can be separated by electrophoresis. 2. Frederick Sanger developed a DNA sequencing method using DNA polymerase and 2, 3 -dideoxynucleotides (nucleotide analogue). DNA synthesis is terminated by incorporation of dideoxynucleotides specifically at A, T, C or G to cause chain-termination and fragments can be separated by electrophoresis.

15 DNA Sequencing BIO Lecture 09/26/2006

16 DNA Sequencing 1) A DNA primer is annealed to the desired DNA. 2) DNA polymerase extends to primer, and labeled nucleotides incorporate in the newly made DNA. 3) 2, 3 -dideoxynucleotides are incorporated and stop DNA synthesis in four test tubes, with each tube containing one dideoxynucleotide such as ddatp, ddgtp, ddctp, and ddttp. For example, ddatp will stop DNA synthesis wherever an adenine nucleotide needs to be inserted. 4) Each resulting strand is a different length, and is separated by electrophoresis.

17 DNA Sequencing BIO Lecture 09/26/2006

18 DNA Sequencing Automated sequencing: many samples and up to bases: 1. Mixing dideoxynucleotides labeled with difference dyes in one tube. 2. Electrophoresis performed in a capillary tube. 3. A laser stimulates the DNA piece, and a recorder sends the information to a computer for analysis.

19 DNA Sequencing BIO Lecture 09/26/2006

20 Molecular Cloning Genetic Phenotype Genetics : Isolate the gene by genetic traits and express the protein to understand the function. Reverse Genetics: Obtain a protein of interest and isolate the gene enncoding for the protein.

21 Molecular Cloning Genetics : Isolate the gene by genetic traits and express the protein to understand the function. Reverse Genetics: Obtain a protein of interest and isolate the gene enncoding for the protein.

22 Protein Methods SDS-PAGE

23 Protein Methods BIO Lecture 09/26/2006

24 Protein Methods BIO Lecture 09/26/2006

25 Protein Methods Edman degradation determines the sequence by modifying the first amino acid of a protein and removing the modified amino acid. This is repeated until sequencing is completed.

26 Protein Methods BIO Lecture 09/26/2006