Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School

Chapter 20: Biotechnology

Ms. Whipple

Brethren Christian High School

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

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

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

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

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

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

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).

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

Fig. 20-3-1Restriction site

DNA

Sticky end

Restriction enzymecuts sugar-phosphatebackbones.

53

35

1


DNA

Sticky end


53

35

1

DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.

2

One possible combination


DNA

Sticky end


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

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

Fig. 20-4-1

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Fig. 20-4-2

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest


Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Fig. 20-4-3

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest


Restrictionsite

Stickyends

ampR gene

TECHNIQUE



Bacteria carryingplasmids

Fig. 20-4-4

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest


Restrictionsite

Stickyends

ampR gene

TECHNIQUE



Bacteria carryingplasmids

RESULTS

Colony carrying non-recombinant plasmidwith intact lacZ gene

One of manybacterialclones

Colony carrying recombinant plasmid with disrupted lacZ gene

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

Fig. 20-5a

Bacterial clones

Recombinantplasmids

Recombinantphage DNA

or

Foreign genomecut up withrestrictionenzyme

(a) Plasmid library (b) Phage library

Phageclones

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

Fig. 20-5b

(c) A library of bacterial artificial chromosome (BAC) clones

Large plasmidLarge insertwith many genes

BACclone

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.

Fig. 20-8a

5

Genomic DNA

TECHNIQUETargetsequence

3

3 5

Fig. 20-8b

Cycle 1yields

2molecules

Denaturation

Annealing

Extension

Primers

Newnucleo-tides

3 5

3

2

5 31

Fig. 20-8c

Cycle 2yields

4molecules

Fig. 20-8d

Cycle 3yields 8

molecules;2 molecules

(in whiteboxes)

match targetsequence

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

Fig. 20-9a

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Longermolecules

Shortermolecules

Gel

AnodeCathode

TECHNIQUE

1

2

Powersource

– +

+–

Fig. 20-9b

RESULTS

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

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

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

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

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

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

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

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

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.

8. How much of the human genome doesn’t code for a protein?

• 98% of the human genome does not directly code for proteins.

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Chapter 20: Biotechnology Ms. Whipple Brethren Christian High School