![Page 1: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/1.jpg)
Chapter 20: Biotechnology
Ms. Whipple
Brethren Christian High School
![Page 2: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/2.jpg)
1. What is Recombinant DNA?
• Recombinant DNA are nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule
• Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes
![Page 3: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/3.jpg)
2. What is Biotechnology and Genetic Engineering? How is Genetic Engineering advancing our society? Give me two examples of genetic
engineering not found in the book.
• Genetic Engineering is the direct manipulation of genes for practical purposes
• DNA technology has revolutionized Biotechnology, the manipulation of organisms or their genetic components to make useful products
• An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes
![Page 4: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/4.jpg)
3. How are Bacteria and Plasmids used for cloning?
• Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids
• Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
• Cloned genes are useful for making copies of a particular gene and/or producing a protein product
![Page 5: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/5.jpg)
3. How are Bacteria and Plasmids used for cloning?
• Gene cloning involves using bacteria to make multiple copies of a gene
• Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell
• Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA
• This results in the production of multiple copies of a single gene
![Page 6: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/6.jpg)
Fig. 20-2a
DNA of chromosome
Cell containing geneof interest
Gene inserted intoplasmid
Plasmid put intobacterial cell
RecombinantDNA (plasmid)
Recombinantbacterium
Bacterialchromosome
Bacterium
Gene ofinterest
Plasmid
2
1
2
![Page 7: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/7.jpg)
Fig. 20-2b
Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest
Gene ofInterest
Protein expressedby gene of interest
Basic research andvarious applications
Copies of gene Protein harvested
Basicresearchon gene
Basicresearchon protein
4
Recombinantbacterium
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growth hor-mone treats stuntedgrowth
3
![Page 8: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/8.jpg)
4. Gene cloning may be used for which two purposes? Give me an example of each.
• Gene cloning is used to make many copies of a gene for research (such as a disease gene) and study or used to make a protein product (such as insulin).
![Page 9: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/9.jpg)
5. How are Restriction Enzymes used for creating Recombinant DNA? Please use the words Restriction Site, Restriction Fragments, Sticky End, and DNA
ligase in your answer.
• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
• A restriction enzyme usually makes many cuts, yielding restriction fragments
• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments
• DNA ligase is an enzyme that seals the bonds between restriction fragments
![Page 10: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/10.jpg)
Fig. 20-3-1Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
53
35
1
![Page 11: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/11.jpg)
Fig. 20-3-2Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
53
35
1
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
One possible combination
![Page 12: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/12.jpg)
Fig. 20-3-3Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
53
35
1
One possible combination
Recombinant DNA molecule
DNA ligaseseals strands.
3
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
![Page 13: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/13.jpg)
6. Briefly describe the steps for gene cloning the B-Globin gene in Hummingbirds.
• Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid:
• The hummingbird genomic DNA and a bacterial plasmid are isolated
• Both are digested with the same restriction enzyme
• The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends
• Some recombinant plasmids now contain hummingbird DNA
• The DNA mixture is added to bacteria that have been genetically engineered to accept it
• The bacteria are plated on a type of agar that selects for the bacteria with recombinant plasmids
• This results in the cloning of many hummingbird DNA fragments, including the β-globin gene
![Page 14: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/14.jpg)
Fig. 20-4-1
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
![Page 15: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/15.jpg)
Fig. 20-4-2
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
![Page 16: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/16.jpg)
Fig. 20-4-3
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carryingplasmids
![Page 17: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/17.jpg)
Fig. 20-4-4
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carryingplasmids
RESULTS
Colony carrying non-recombinant plasmidwith intact lacZ gene
One of manybacterialclones
Colony carrying recombinant plasmid with disrupted lacZ gene
![Page 18: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/18.jpg)
7. What is the Genomic Library? How is this used in research?
• A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome
• A genomic library that is made using bacteriophages is stored as a collection of phage clones
![Page 19: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/19.jpg)
Fig. 20-5a
Bacterial clones
Recombinantplasmids
Recombinantphage DNA
or
Foreign genomecut up withrestrictionenzyme
(a) Plasmid library (b) Phage library
Phageclones
![Page 20: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/20.jpg)
8. What is a Bacterial Artificial Chromosome? What is the advantage of using this for research?
• A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert
• BACs are another type of vector used in DNA library construction
![Page 21: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/21.jpg)
Fig. 20-5b
(c) A library of bacterial artificial chromosome (BAC) clones
Large plasmidLarge insertwith many genes
BACclone
![Page 22: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/22.jpg)
1. What is PCR? What is it used for?
• The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA
• A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules
• This is a very useful method if the DNA source is very limited or impure as it can make many many copies of one segment in a short amount of time.
![Page 23: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/23.jpg)
Fig. 20-8a
5
Genomic DNA
TECHNIQUETargetsequence
3
3 5
![Page 24: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/24.jpg)
Fig. 20-8b
Cycle 1yields
2molecules
Denaturation
Annealing
Extension
Primers
Newnucleo-tides
3 5
3
2
5 31
![Page 25: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/25.jpg)
Fig. 20-8c
Cycle 2yields
4molecules
![Page 26: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/26.jpg)
Fig. 20-8d
Cycle 3yields 8
molecules;2 molecules
(in whiteboxes)
match targetsequence
![Page 27: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/27.jpg)
2. What is Gel Electrophoresis? How is it used for research?
• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
• This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size
• A current is applied that causes charged molecules to move through the gel
• Molecules are sorted into “bands” by their size
• In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis
• Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene
• The procedure is also used to prepare pure samples of individual fragments
![Page 28: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/28.jpg)
Fig. 20-9a
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
1
2
Powersource
– +
+–
![Page 29: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/29.jpg)
Fig. 20-9b
RESULTS
![Page 30: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/30.jpg)
Fig. 20-10
Normalallele
Sickle-cellallele
Largefragment
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
201 bp175 bp
376 bp
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
Normal -globin allele
Sickle-cell mutant -globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp175 bp
DdeIDdeI
DdeI DdeI DdeI DdeI
![Page 31: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/31.jpg)
3. Briefly describe the Southern blotting method? What is this used for?
• A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization
• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel
![Page 32: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/32.jpg)
Fig. 20-11a
TECHNIQUE
Nitrocellulosemembrane (blot)
Restrictionfragments
Alkalinesolution
DNA transfer (blotting)
Sponge
Gel
Heavyweight
Papertowels
Preparation of restriction fragments Gel electrophoresis
I II IIIDNA + restriction enzyme
III HeterozygoteII Sickle-cellallele
I Normal-globinallele
1 32
![Page 33: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/33.jpg)
Fig. 20-11b
I II IIII II III
Film overblot
Probe detectionHybridization with radioactive probe
Fragment fromsickle-cell-globin allele
Fragment fromnormal -globin allele
Probe base-pairswith fragments
Nitrocellulose blot
4 5
Radioactively labeledprobe for -globin gene
![Page 34: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/34.jpg)
4. What two methods can be used to study gene changes in embryonic development? Briefly describe them?
• Changes in the expression of a gene during embryonic development can be tested using
• Northern blotting combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane
• Identification of mRNA at a particular developmental stage suggests protein function at that stage
• Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive
• Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest
• The products are run on a gel and the mRNA of interest identified
• Both methods are used to compare mRNA from different developmental stages
![Page 35: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/35.jpg)
5. What is the purpose and method of a DNA microarray assay?
• Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays
• DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions
![Page 36: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/36.jpg)
Fig. 20-15
TECHNIQUE
Isolate mRNA.
Make cDNA by reversetranscription, usingfluorescently labelednucleotides.
Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray.
Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.
Tissue sample
mRNA molecules
Labeled cDNA molecules(single strands)
DNA fragmentsrepresentingspecific genes
DNA microarraywith 2,400human genes
DNA microarray
1
2
3
4
![Page 37: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/37.jpg)
6. What is the purpose and method of in vitro mutagenesis and RNA interference?
• One way to determine function is to disable the gene and observe the consequences
• Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function
• When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype
• Gene expression can also be silenced using RNA interference (RNAi)
• Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA
![Page 38: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/38.jpg)
7. When studying humans, what is the purpose of looking for a single nucleotide polymorphism? How does this aid us in finding and tracking
human genetic diseases?
• A single nucleotide polymorphism (SNP) is a single base pair site where a variation is found in at least 1% of the population.
• Once a gene (especially disease genes) has been found to have a SNP shared by affected people and not unaffected people, the gene is sequenced.
• The SNP and disease causing gene will most likely be inherited together because they are close to each other on the genome. Therefore, the SNP can be used as a marker for the disease.
• Doctors can perform a sensitive microarray analysis to look for SNPs or by PCR.
![Page 39: Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School](https://reader030.vdocument.in/reader030/viewer/2022032705/56649d875503460f94a6c626/html5/thumbnails/39.jpg)
8. How much of the human genome doesn’t code for a protein?
• 98% of the human genome does not directly code for proteins.