Gene Splicing and Restriction Maps

Transcription

1 Gene Splicing and Restriction Maps

2 Bacteria have a large circular chromosome as well as many smaller circular structures called plasmids. These plasmids are an important tool in gene splicing. 1 µm

3 Bacteria have a large circular chromosome as well as many smaller circular structures called plasmids. These plasmids are an important tool in gene splicing. 1 µm

4 Gene splicing to produce insulin 1. Human DNA is cut into fragments using a restriction enzyme.

5 Gene splicing to produce insulin Human cell The restriction enzyme must be specially chosen to cut on either side of the insulin gene. insulin gene DNA 1. Human DNA is cut into fragments using a restriction enzyme. Many fragments will be made, but one will have the insulin gene.

6 Gene splicing to produce insulin 1. Human DNA is cut into fragments using a restriction enzyme. Many fragments will be made, but one will have the insulin gene. 2. Plasmids are cut with the same restriction enzyme as in step1.

7 Gene splicing to produce insulin complimentary ends Using the same restriction enzyme means that the plasmids and the fragments have complimentary ends. The antibiotic resistance gene will be important in a later process. antibiotic resistance gene 1. Human DNA is cut into fragments using a restriction enzyme. Many fragments will be made, but one will have the insulin gene. 2. Plasmids are cut with the same restriction enzyme as in step1. The plasmids also contains an antibiotic resistance gene.

8 Gene splicing to produce insulin 3. Mix the DNA fragments, the cut plasmids and DNA ligase, to produce r e c o m b i n a n t D N A plasmids.

9 Some of these recombinant plasmids will contain the insulin gene, and some will not. 3. Mix the DNA fragments, the cut plasmids and DNA ligase, to produce r e c o m b i n a n t D N A plasmids.

10 Gene splicing to produce insulin 4. Through transformation, recombinant plasmids enter bacterial cells. As the bacteria divide, billions of copies of the recombinant plasmids are made. Some of these bacteria will made insulin.

11 Gene splicing to produce insulin 4. Through transformation, recombinant plasmids enter bacterial cells. As the bacteria divide, billions of copies of the recombinant plasmids are made. Some of these bacteria will make made insulin.

12 Gene splicing to produce insulin Hybridization requires these colonies to be antibiotic resistant. 5. A process called hybridization is used to identify the bacterial colonies that produce insulin. These colonies will be isolated and given the optimal conditions needed for producing large quantities of insulin.

13 Choosing the proper restriction enzyme for a given gene and plasmid requires a detailed map of the restriction sites found on each plasmid. For example, if EcoR1 will cut on either side of the insulin gene, we must find a plasmid that contains the EcoR1 restriction site. Human cell The restriction enzyme must be specially chosen to cut on either side of the insulin gene. insulin gene DNA

14 Do Q #5, 6 and 8 pg. 375 Take your own notes on Hybridization (Figure 11 on pg. 374)

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16 Choosing the proper restriction enzyme for a given gene and plasmid requires a detailed map of the restriction sites found on each plasmid. For example, if EcoR1 will cut on either side of the insulin gene, we must find a plasmid that contains the EcoR1 restriction site. Researchers expose plasmids to different restriction enzymes and then measure the length of the pieces produced. They use this data to construct a restriction map for the plasmid.

17 Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and Table 1: Results of Plasmid X Digestion with EcoR1 and undigested (bp) digested with EcoR1 (bp) digested with (bp) digested with EcoR1 and (bp) EcoR1 Plasmid X 1400bp

18 Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and Table 1: Results of Plasmid X Digestion with EcoR1 and undigested (bp) digested with EcoR1 (bp) digested with (bp) digested with EcoR1 and (bp) bp 800 bp Plasmid X

19 Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and Table 1: Results of Plasmid X Digestion with EcoR1 and undigested (bp) digested with EcoR1 (bp) digested with (bp) digested with EcoR1 and (bp) bp bp 100 bp

20 Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and EcoR1 bp Plasmid X 1400bp 800 bp Plasmid X 700 bp bp 100 bp

21 Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and EcoR1 bp 800 bp 700 bp bp 100 bp

22 Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and If the recognition site for EcoR1 was at the same location as the recognition site, you would expect the following fragments. actual results EcoR1 expected results digested with EcoR1 and (bp) bp bp digested with EcoR1 and (bp) bp bp 100 bp

23 Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and If the recognition site for EcoR1 was 100 bp clockwise from the recognition site, you would expect the following fragments. actual results EcoR1 expected results digested with EcoR1 and (bp) bp 100 bp bp 500 bp digested with EcoR1 and (bp) bp bp 100 bp

24 Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and If the recognition site for EcoR1 was 100 bp counterclockwise from the recognition site, you would expect the following fragments. actual results EcoR1 expected results digested with EcoR1 and (bp) bp 700 bp 100 bp 100 bp bp 500 bp digested with EcoR1 and (bp) bp bp 100 bp

25 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and expose Plasmid Y to only EcoR1 (single digestion) expose Plasmid Y to only (single digestion) expose Plasmid Y to both EcoR1 and (double digestion) Here are the results! Plasmid Y

26 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and Table 2: Results of a Restriction Fragment Digestion of Plasmid Y EcoR1 (bp) (bp) EcoR1 and (bp) Plasmid Y

27 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and Table 2: Results of a Restriction Fragment Digestion of Plasmid Y EcoR1 (bp) (bp) EcoR1 and (bp) EcoR bp 1200 bp Plasmid Y EcoR1

28 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and Table 2: Results of a Restriction Fragment Digestion of Plasmid Y EcoR1 (bp) (bp) EcoR1 and (bp) bp 1200 bp Plasmid Y

29 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and Table 2: Results of a Restriction Fragment Digestion of Plasmid Y EcoR1 (bp) (bp) EcoR1 and (bp) bp 400 bp 200 bp 200 bp

30 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and EcoR1 bp bp 1200 bp Plasmid Y 1200 bp Plasmid Y EcoR bp 400 bp 200 bp 200 bp

31 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and EcoR1 bp bp 1200 bp 1200 bp EcoR bp 400 bp 200 bp 200 bp

32 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and If the recognition sites for EcoR1 were at the same locations as the recognition sites, you would expect the following fragments. actual results EcoR1 expected results digested with EcoR1 and (bp) digested with EcoR1 and (bp) bp bp 1200 EcoR bp 400 bp 200 bp 200 bp

33 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and If the recognition sites for EcoR1 were 200 bp clockwise from the recognition sites, you would expect the following fragments. actual results EcoR1 digested with EcoR1 and (bp) bp bp EcoR bp 400 bp 200 bp 200 bp

34 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and If the recognition sites for EcoR1 were 200 bp clockwise from the recognition sites, you would expect the following fragments. actual results digested with EcoR1 and (bp) bp 1200 bp 1000 bp EcoR1 200 bp bp 400 bp 200 bp bp EcoR bp 400 bp 200 bp 200 bp

35 Sample Problem 2: Construct a restriction map for Plasmid Y for EcoR1 and If the recognition sites for EcoR1 were 200 bp clockwise from the recognition sites, you would expect the following fragments. actual results EcoR1 expected results digested with EcoR1 and (bp) bp 200 bp 200 bp 400 bp digested with EcoR1 and (bp) EcoR bp 400 bp 200 bp 200 bp

36 Choosing the proper restriction enzyme for a given gene and plasmid requires a detailed map of the restriction sites found on each plasmid. For example, if EcoR1 will cut on either side of the insulin gene, we must find a plasmid that contains the EcoR1 restriction site. Researchers expose plasmids to different restriction enzymes and then measure the length of the pieces produced. They use this data to construct a restriction map for the plasmid.

37 Researchers expose plasmids to different restriction enzymes and then measure the length of the pieces produced. They use this data to construct a restriction map for the plasmid. Sample Problem 1: Construct a restriction map for Plasmid X for EcoR1 and expose Plasmid X to only EcoR1 (single digestion) expose Plasmid X to only (single digestion) expose Plasmid X to both EcoR1 and (double digestion) The length of the pieces are determined using a technique called gel electrophoresis. Plasmid X Here are the results!