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DNA Technology & Genomics

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1 DNA Technology & Genomics
Chapter 6 Biotechnology: DNA Technology & Genomics 1

2 The BIG Questions… How can we use our knowledge of DNA to:
diagnose disease or defect? cure disease or defect? change/improve organisms? What are the techniques & applications of biotechnology? Biotechnology The use of living organisms (or substances from living organisms) for practical purposes 2

3 Biotechnology Use of organisms to create a desired product is not new
humans have been doing this for thousands of years Alcohol fermentation (for brewing beer and food preservation) INDIRECTLY manipulating DNA 3

4 Biotechnology Agriculture Plant and animal breeding
Selective breeding – altered the genomes of crops by breeding them with other plants. INDIRECTLY manipulating DNA 4

5 Biotechnology today Genetic Engineering Direct manipulation of DNA
if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with this chapter is a survey of those tools… 5

6 Bioengineering Tool kit
Basic Tools restriction enzymes ligase plasmids / cloning DNA databases / probes Advanced Tools PCR DNA sequencing gel electrophoresis Southern blotting microarrays

7 Isolating DNA Before DNA can be manipulated, it needs to be isolated from cells. 1. Cell membranes are disrupted use a detergent Salt used to neutralize lipids 2. DNA precipitation ethanol used to dehydrate and aggregate DNA Salt used to neutralize phosphate groups in DNA 3. DNA isolation / storage e.g. Sharks do not get sick or get cancer. Scientists have determined the protein and the gene that is responsible for immunity in sharks.

8 DNA Isolation

9 1. Cut DNA To study the functions of individual genes, molecular biologists will cut desired genes out of a genome place the gene into bacterial plasmid produce recombinant DNA

10 1. Cut DNA treat isolated DNA with restriction enzymes
restriction endonucleases Evolved in bacteria Immune System protection against viruses & other bacteria

11 Restriction enzymes - Action
cut DNA at specific sequences Restriction (recognition) site Are usually 4-8 bp in length produces protruding ends sticky ends that contain DNA nucleotides that lack complementary bases Some do not produce protruding ends blunt ends CTGAATTCCG GACTTAAGGC CTG|AATTCCG GACTTAA|GGC 11

12 Restriction Enzymes Many different enzymes
named after organism they are found in EcoRI, HindIII, BamHI, SmaI

13 Restriction Enzyme Cutting
sticky ends – enzyme digests (cuts) to make overhangs EcoRI 5’ G A A T T C 3’ 3’ C T T A A G 5’ 5’ G 3’ 5’ A A T T C 3’ 3’ C T T A A 5’ 3’ G 5’ 5’ overhang

14 Restriction Enzyme Cutting
PstI 5’ C T G C A G 3’ 3’ G A C G T C 5’ 5’ C T G C A 3’ 5’ G 3’ 3’ G 5’ 3’ A C G T C 5’ 3’ overhang

15 2. Paste DNA Sticky ends allow: DNA Ligase
H bonds between complementary bases to anneal DNA Ligase enzyme “seals” strands bonds sugar-phosphate backbone together Condensation reaction 15

16 DNA Ligase T4 DNA ligase originated in T4 bacteriophages
used to chemically join two blunt ends of DNA together

17 Biotech use of restriction enzymes
GAATTC CTTAAG DNA Restriction enzyme cuts the DNA Sticky ends (complementary single-stranded DNA tails) AATTC G AATTC G G CTTAA G CTTAA Add DNA from another source cut with same restriction enzyme AATTC G G AATTC CTTAA G DNA ligase joins the strands. 17 Recombinant DNA molecule GAATTC CTTAAG

18 Exercise 1

19 Cut, Paste, Copy, Find… Word processing metaphor… 1. cut 2. paste
Isolate desired DNA restriction enzymes 2. paste ligase 3. copy plasmids bacteria transformation PCR 4. find Southern blotting / probes 19

20 Remember… Prokaryotic genomes (e.g. bacteria) contain Chromosome
Plasmids Chromosome Plasmid

21 Plasmid pBR322 Plasmids Contain “accessory genes”
Antibiotic resistance Can carry and express foreign genes Plasmids are vectors Vehicles by which DNA can be introduced into host cells

22 Plasmid Restriction Maps
Shows the location of cleavage sites for many different enzymes These maps are used like road maps to the DNA molecule Numbers beside restriction enzyme indicates at which base pair the DNA is cut by that particular enzyme

23 Sample Problem: Determine the size and number of fragments that would be produced if the plasmid was digested with: EcoRV and Pvu II? 2 cuts, therefore 2 fragments Between sites: =1879 bp Rest: =2482 bp

24 Why bacteria? Plasmid uptake is easy Cheap
Bacterium Bacterial chromosome Plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest DNA of chromosome Recombinate bacterium Protein harvested Basic research on protein Copies of gene Basic research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth Protein expressed by gene of interest 3 Why bacteria? Plasmid uptake is easy Cheap Reproduce rapidly and frequently Produce multiple copies of the recombinant DNA and protein in a short amount of time Recombinant DNA and proteins are kept in large storage database to be used later

25 Actin Protein Unnamed Protein
Protein Databases Actin Protein Unnamed Protein

26 Review: Transformation vs. Recombination
the introduction of foreign DNA (usually a plasmid) into a bacterial cell Recombination Fragment of DNA composed of sequences originating from at least two different sources

27 3. Copying (Cloning) Figure 20.4 TECHNIQUE
In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes -galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and three 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. Sticky ends Human DNA fragments Human cell Gene of interest Bacterial cell ampR gene (ampicillin resistance) Bacterial plasmid Restriction site Recombinant DNA plasmids lacZ gene (lactose breakdown) Figure 20.4 3. Copying (Cloning) 1 Isolate plasmid DNA and human DNA. 2 Cut both DNA samples with the same restriction enzyme 3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids.

28 Introduce the DNA into bacterial cells (transoformation).
RESULTS Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest. Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone Colony carrying recombinant plasmid with disrupted lacZ gene Recombinant bacteria 4 Introduce the DNA into bacterial cells (transoformation). 5 Plate the bacteria on agar containing ampicillin and X-gal (lactose). Incubate until colonies grow.

29 How do we know if it worked?
We know cloning worked if: Transformation has occurred i.e. there has been plasmid uptake into the bacteria Recombination has occurred i.e. foreign genes have been inserted into the bacterial plasmid

30 Selecting for successful transformation
Antibiotic resistance genes as a selectable marker Plasmid has both “added” gene & antibiotic resistance gene selection How do we know what’s the right combination of genes on a plasmid? Trail and error research work. selectable markers high copy rate convenient restriction sites There are companies that still develop plasmids, patent them & sell them. Biotech companies (ex. New England BioLabs) 30

31 Amp Selection If bacteria don’t pick up plasmid
they will not have antibiotic resistance die on antibiotic (amp) plates If bacteria pick up plasmid survive on antibiotic (amp) plates We have selected only those colonies that have undergone successful transformation

32 only transformed bacteria grow
Amp Selection Ampicillin is the selecting agent only transformed bacteria grow all bacteria grow Some bacteria on the LB/amp plate can have bacteria with recombinant plasmids and bacteria with non-recombinant plasmids LB plate LB/amp plate 32

33 Screening for successful recombination
Transformed colonies have both recombinant and non-recombinant plasmids recombinant non-recombinant

34 LacZ gene codes for β galactosidase
LacZ Screening LacZ gene codes for β galactosidase Lactose (or X-gal) is our screening agent plasmid amp resistance LacZ gene restriction sites lactose  blue color recombinant plasmid amp resistance broken LacZ gene lactose  white color X inserted gene of interest origin of replication EcoRI all in LacZ gene BamHI HindIII Colour tells us to distinguish between 34

35 LacZ screening system We want these!!
Make sure inserted plasmid is recombinant plasmid LacZ gene on plasmid produces digestive enzyme lactose(X-gal)  blue blue colonies insert foreign DNA into LacZ gene breaks gene lactose (X-gal)  blue white colonies white bacterial colonies have recombinant plasmid X X We want these!! 35

36 Amp selection & LacZ screening
gene of interest LacZ gene - amp resistance LB/amp LB/amp/Xgal 36

37     Cut, Paste, Copy, Find… Word processing metaphor… cut paste
restriction enzymes paste ligase copy plasmids bacteria transformation PCR find Southern blotting / probes 37

38 4. Find White colonies could have recombinant plasmids
desired and undesired genes How do you find the conony with the gene of interest?

39 But how do we find colony with our gene of interest in it?
recombinant plasmids inserted into bacteria gene of interest human genome = 3 billion bases fragments are cut to ~5000 bases therefore ~ 600,000 fragments per cell. But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes. bacterial colonies (clones) grown on LB/amp/Xgal petri plates 39

40 Locating your gene of interest
DNA hybridization find gene in bacterial colony using a probe short, single stranded DNA molecule complementary to part of gene of interest tagged with radioactive P32 or fluorescence heat treat genomic DNA unwinds (denatures) strands DNA hybridization between probe & denatured DNA 5’ 3’ labeled probe genomic DNA G A T C

41 Hybridization 4 1 2 3 Locate Cloning expose film
locate colony on plate from film Cloning - plate with bacterial colonies carrying recombinant plasmids 1 plate plate + filter film 2 Replicate plate press filter paper onto plate to take sample of cells from every colony Hybridization - heat filter paper to denature DNA - wash filter paper with radioactive probe which will only attach to gene of interest filter 3

42 Classwork/Homework Pg. 281 #1-3 Pg. 282 #6,7,10 Pg. 287 #16,18
Pg. 291 #2-7,10,11(b,c),15,16(a,b),17,19 Pg. 295 #4,5

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