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Chapter 3 Recombinant DNA Technology (genetic engineering)

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1 Chapter 3 Recombinant DNA Technology (genetic engineering)

2 Enzymes that cut and paste DNA Restriction enzymes cut DNA at specific base sequences called restriction sites

3 Enzymes that cut DNA are called restriction enzymes

4 Enzyme DNA ligase enzyme pastes cut ends back together

5 Cloning: the introduction of new or foreign genes into plasmids and other vectors This is when scientists take control of the natural processes that the bacteria have evolved to promote exchange of genes between individuals of the same or different species Circular extrachromosomal DNA found commonly in bacteria Plasmid DNA is replicated at same time chromosomal DNA is replicated Used to pass genes back and forth between different bacteria A T C G

6 Bacterial cells are efficient ways to produce lots of copies of a foreign gene introduced into a plasmid

7 Cloning Plasmids serve as cloning vectors Tumor-inducing DNA (Ti plasmid) contains 8 tumor- inducing genes Use this plasmid to introduce a new gene into a plant chromosome transformation

8 Concerns about cloning What might happen if cloned bacteria were to leave the lab and transfer their genes to other bacteria or even humans? –E. coli was initially the most common host for these cloned genes –Benefits and hazards discussed in 1975 at a meeting –National Institutes of Health (NIH) formed the Recombinant DNA Advisory Committee (RAC) –Guidelines established for recombinant DNA research by scientific community

9 Review of molecular biologists toolbox Plasmids Restriction enzymes DNA ligase Host bacterial cells to replicate plasmids

10 Recombinant DNA technology has become a way for geneticists to express genes from other organisms in bacteria Human insulin gene was cloned into a bacterial plasmid and expressed (gene mRNA protein) in a bacterium in 1977. -Cheap and pure source of insulin Humulin growth hormone was first recombinant DNA product to be approved by FDA in 1992 Currently over 100 products on market produced by recombinant techniques

11 Multiple cloning site inside lacZ gene (restriction site for insertion site for foreign gene) Foreign DNA Section of foreign DNA with gene of interest Plasmid cloning vector Mix plasmid and foreign DNA together with restriction enzyme and DNA ligase Restriction enzyme DNA ligase Restriction site P O This plasmid has the lacZ gene inserted

12 Plasmid cloning vector Extra-chromosomal DNA carried by bacterial cell Ampicillin resistance gene amp R (selective marker) Multiple cloning site inside lacZ gene (restriction site for insertion site for foreign gene) lacZ gene with promoter (used to switch in expression of foreign gene when inside a bacterial host cell) Foreign gene inserted

13 Insert plasmid into host bacterial cell for replication Bacterial cell chromosome

14 Cultivate host cell to replicate and produce many copies of foreign gene Bacterial cell

15 Detecting cells that have foreign gene inserted in lacZ gene on plasmid Need some way to check to see that foreign gene was inserted into the plasmid so when you cultivate the cell, you know you are producing more copies of foreign gene

16 Switching on expression of foreign gene during cultivation of host bacterial cell xGal (lactose) RNA polymerase colored product enzyme mRNA If no foreign gene inserted into restriction site, then blue colored product is produced plasmid chromosome No foreign gene inserted

17 RNA polymerase no product (no color) no enzyme mRNA xGal If foreign gene is inserted into restriction site, then no colored product is produced

18 Plating cells on agar surface to promote colony formation Semisolid nutrient medium for bacterial cell to replicate to produce many daughter cells to form a visible colony Visible colony of identical cells Medium contains ampicillin to allow only the bacterial cells that contain plasmid with amp R gene to grow

19 Cloning (restriction sites)


21 Types of vectors Bacterial plasmid bacteriophage cosmids bacterial artificial chromosome yeast artificial chromosome 6-12 25 35 300 200-1000 Maximum insert size (kilobases or kb [1000bp])

22 Practical Features of DNA Cloning Vectors (Plasmids) origin of replication (ori) multiple cloning sites (MCS) or restriction sites selectable markers RNA polymerase promoter sequences DNA sequencing primer sequences oriamp R MCS lacZ gene If plasmid picks up a foreign piece of DNA at the MCS, then the lacZ gene is non-functional Allows bacteria with this plasmid to grow in presence of ampicillin antibiotic

23 You can use plasmids to create a clone library Purpose: To distribute different sections of a DNA molecule or chromosome into a vector that allows the genes contained in the section to be characterized

24 Making a genomic library Plate out to form colonies

25 Screening clones for plasmids that picked up foreign DNA fragment

26 What if you know a part of the base sequence of the gene you are looking for? The Human Genome Project has given us this information for all the genes in our chromosomes

27 stopped

28 Polymerase chain reaction (PCR) Has revolutionized molecular biology and biotechnology. Most useful when you know at least some of the base sequence of the gene you are interested in Only need to know a sequence containing 10-20 base pairs in a gene that may contain thousands of base pairs

29 Design primers that specifically target sequences at the ends of the foreign gene Foreign gene Plasmid

30 Polymerase Chain Reaction (PCR) Much more rapid approach to cloning than making or screening clone libraries. Makes lots of copies of foreign gene that is then inserted into plasmid Need to know part of sequence of gene

31 Cloning a gene by PCR Uses a restriction enzyme that recognizes A-T restriction site for cutting T vector for insertion of gene

32 Host bacterial cell T-plasmid vector containing same foreign gene

33 Now, every transformed bacterial cell that picks up the plasmid contains the same fragment gene of foreign DNA

34 How do you recover foreign DNA fragment containing gene of interest? Pellet cells from culture medium Resuspend cells in solution that breaks up lyses cells to release DNA Separate host cell DNA from plasmid DNA by electrophoresis DNA bands

35 Separating DNA fragments produced by treatment with restriction enzymes Agarose gel electrophoresis

36 Each band represents a different size fragment created by cutting the chromosome with a restriction enzyme Different lanes on gel contain fragments of same DNA cut with different restriction enzymes When you separate DNA fragments on a gel it is called a Southern gel Restriction mapping

37 Restriction Mapping Fragment of chromosome This is the technique used for DNA fingerprinting

38 Gels that show genes that are being expressed Gels that reveal mRNA or other types of RNA are called Northern gels

39 Testing all genes expressed in a tissue quickly using microarray or gene chip

40 Each spot contains millions of copies of short, single-stranded DNA-a different gene in each spot Gene 1 Gene 2 AACTCAACTC ACCTCACCTC UGGAGUGGAG

41 Computer scans chip and provides a printout of which genes were expressed

42 Bioinformatics Database manipulation of DNA sequence information Application of computer science and information technology to help understand biological processes Use of computers to relate gene sequence to protein structure and function

43 Example of bioinformatics Alignment of overlapping sequences used to assemble sequence of large pieces of DNA (chromosomes)

44 Using Bioinformatics GenBank-a library of base sequences that have been catalogued – useful for matching your sequences from your clone library with sequences found and deposited by others previously –go to blastn –type in AATAAGAACCAGGAGTGGA –BLAST finds the match to your sequence to be the gene for early-onset breast cancer, BRCA-1 each unique sequence is assigned an accession number to make it easy for scientists to refer back to that sequence

45 Comparing the human and mouse genome

46 More things you can do –search Omim database –type in a word for a disease then search the database provides you with a list of diabetes- related genes click on one-it provides you with all types of information on these genes click on gene map –click on IDDM1 »click on 6p21.3 »it shows you the locus on the chromosome where the gene resides (find 222100) »click on 222100-it verifies that you have located the gene of interest

47 Search for a gene you are interested in –lists different metabolism along left –at top click here takes you to all the chromosomes –click on chromosome 7 gives you more info on the genes on that chromosome –shows you where the genes for different diseases are located on that chromosome.

48 Summary Restriction sites and enzymes Cloning vectors (plasmids) Inserting foreign genes in plasmids Hosts cells for replicating plasmids (bacteria) Clone libraries, cDNA libraries Screening for recombinant plasmids Polymerase chain reaction (PCR) Reverse transcription PCR for detecting mRNA Separating DNA fragments on gels Gene chips Bioinformatics

49 Some companies doing this work want to patent the sequences of fragments of our DNA Cost of bringing a new drug (protein) to market is about $500 million –Takes 5-8 years to do this They see opportunities to turn this into a money-making endeavor A patent gives legal exclusive right to control use of sequences contained within fragment

50 Patents Companies want assurances that after investing their resources to get a product approved for use that another company cant come in and make $$ without such an investment Since 1980, the U.S. Patent Office has awarded patents on more than 20,000 gene sequences

51 Patent process 3 categories –products or composition of matter –methods of use –manufacturing processes Conditions that must be met to receive a patent –must be new (not previously published or described) –must be useful –not obvious to one skilled in the field

52 Elements of a patent application Description of technical field to which invention applies Description of problems to be solved and prior art How the invention improves upon prior art Summary enumerating fundamental components of invention Description of invention and indispensable steps for constructing invention

53 Elements of a patent application Claims that outline the elements to be protected by law. –A claim cannot be so broad that it infringes upon prior art –A claim should not be so narrowly focused that the applicant could risk losing property claims Patent attorneys are skilled in preparing patent application Upon review of application by U.S. Patent Office, a decision will be made whether a new patent is justified-if so, a patent no. is assigned

54 New Patent Issues Sequences may or may not encode a gene Sequence may control regulation of nearby genes. Many scientists believe patenting should be reserved for the new technology used to discover genes and their functions and their application rather than the sequence. Is it ethical to patent a sequence? What are the possible consequences?

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