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 gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA (plasmid) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Plasmid 2 1 2

Fig. 20-2b Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein 4 Recombinant bacterium 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 hor- mone treats stunted growth 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 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones

Fig Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2 One possible combination

Fig Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones One possible combination Recombinant DNA molecule DNA ligase seals strands. 3 DNA fragment added from another molecule cut 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 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE

Fig Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid

Fig Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids

Fig Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene One of many bacterial clones 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 Recombinant plasmids Recombinant phage DNA or Foreign genome cut up with restriction enzyme (a) Plasmid library(b) Phage library Phage clones

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 plasmid Large insert with many genes BAC clone

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 TECHNIQUE Target sequence 3 3 5

Fig. 20-8b Cycle 1 yields 2 molecules Denaturation Annealing Extension Primers New nucleo- tides

Fig. 20-8c Cycle 2 yields 4 molecules

Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

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 of DNA mol- ecules of different sizes Power source Longer molecules Shorter molecules Gel Anode Cathode TECHNIQUE 1 2 Power source – + + –

Fig. 20-9b RESULTS

Fig Normal allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of  -globin gene Normal  -globin allele Sickle-cell mutant  -globin allele Dde I Large fragment 376 bp 201 bp 175 bp Dde I

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 a TECHNIQUE Nitrocellulose membrane (blot) Restriction fragments Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragmentsGel electrophoresis I II III DNA + restriction enzyme III Heterozygote II Sickle-cell allele I Normal  -globin allele 1 32

Fig b I II III Film over blot Probe detectionHybridization with radioactive probe Fragment from sickle-cell  -globin allele Fragment from normal  -globin allele Probe base-pairs with fragments Nitrocellulose blot 4 5 Radioactively labeled probe 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 TECHNIQUE Isolate mRNA. Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA fragments representing specific genes DNA microarray with 2,400 human genes DNA microarray

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.