Chapter 20 Biotechnology

Chapter 20 Biotechnology

Manipulation of DNA In 2007, the first entire human genome had been sequenced. The ability to sequence an organisms genomes were made possible by advances in biotechnology, (the manipulation of organisms or their components to make useful products) and genetic engineering (the direct manipulation of genes for practical purposes). Recombinant DNA, DNA molecules formed when segments of DNA from two different sources (often different species) are combined in vitro.

Concept 20.1 DNA cloning yields multiple copies of a gene or other DNA segments DNA cloning can be used to amplify DNA sequences of interest Restriction enzymes can be used to insert foreign DNA into bacterial plasmids which can then be transformed into bacteria to produce recombinant bacteria

DNA Cloning Recall: The E. coli chromosome is a large circular molecule of DNA. In addition, bacteria may also contain plasmids, small circular DNA molecules. In gene cloning, multiple copies of a single gene are produced. To clone DNA, plasmids can be isolated from a bacterial cell and then foreign DNA can be inserted to create a recombinant DNA molecule. This is inserted back into the bacterium to generate a recombinant bacterium which will undergo cell division to produce more recombinant bacteria.

Applications of DNA Cloning A bacterial plasmid is a cloning vector a DNA molecule that can carry foreign DNA into a host cell and replicate there. Foreign DNA can be inserted into the cloning vector to generate a recombinant plasmid Two purposes of gene cloning: Make many copies of a gene Produce protein product Both of these can be isolated and harvested for use in research. They also have applications purposes in many industries E.g. agriculture, medicine

Bacterium 1 Gene inserted into plasmid Cell containing gene of interest Bacterial chromosome Plasmid Recombinant DNA (plasmid) 2 Gene of interest Plasmid put into bacterial cell DNA of chromosome Recombinant bacterium 3 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 Copies of gene Protein harvested Basic research on gene 4 Basic research and various applications Basic research on protein 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

Restriction Enzymes Restriction enzymes enzymes that cut DNA molecules at a limited number of specific locations based on sequence. Restriction enzymes are able to recognize restriction sites, which are short specific sequences of DNA. Restriction enzymes can cut both DNA strands within the restriction site, most of which are symmetrical The pieces of DNA produced are known as restriction fragments Methylation of adenines or cytosines can protect bacterial DNA from the restriction enzymes

Making Recombinant DNA Restriction enzymes cleave the sugar phosphate backbone in both strands of DNA, usually leaving a uneven end. This uneven end produces one longer single-stranded end, which is known as a sticky end. This can be hydrogen bonded with complementary single-stranded DNA. Ligase can then catalyze the formation of new bonds in the sugar phosphate backbone. This method can be used to insert DNA into an existing molecule, forming recombinant DNA.

Restriction site DNA 5 3 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 3 DNA ligase seals strands. One possible combination Recombinant DNA molecule

Cloning Genes in Bacterial Plasmids Cloning of a hummingbird gene: 1. Isolate bacterial plasmid and isolate hummingbird DNA. Bacterial plasmid contains the genes amp R and lacz. 2. Both samples of DNA are then digested with the same restriction enzyme. The restriction enzyme chosen only has one site in the bacterial plasmid which is within the lacz gene. 3. The fragments generated will then base pair their complementary sticky ends and DNA ligase can bond the sugar phosphate backbones.

Cloning Genes in Bacterial Plasmids Cloning continued 4. The DNA mixture is then added to a bacterial culture with bacteria that have a mutation in their lacz gene. 5. Bacteria is then plated on nutrient agar with ampicillin and X-gal, which will select for colonies that have taken in recombinant plasmid. 6. White colonies that grow on this medium will have taken in recombinant plasmid. The recombinant plasmid will contain different fragments of hummingbird DNA.

Selecting for Recombinant Bacteria The medium contains ampicillin and X-gal to identify recombinant bacteria The amp R gene provides ampicillin resistance. Therefore, only bacteria with the amp R gene will survive on medium with ampicillin. This verifies the presence of the plasmid in the bacteria. Only bacteria with functioning β-galactosidase will produce a blue colonies due to cleavage of X-gal by β-galactosidase, which produces a blue product. Therefore, only white colonies will have a recombinant plasmid within (hummingbird DNA will have inserted into the lacz gene of the plasmid).

TECHNIQUE Bacterial cell lacz gene Hummingbird cell amp R gene Bacterial plasmid Restriction site Sticky ends Gene of interest Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lacz gene Colony carrying recombinant plasmid with disrupted lacz gene One of many bacterial clones

Storing Cloned Genes in DNA Libraries The cloning procedure produces colonies of recombinant bacteria that contain foreign genomic DNA. A genomic library is the complete set of plasmidcontaining clones, each carrying copies of a particular segment from the initial genome. A genomic library that is made using bacteria is the collection of recombinant vector clones. A genomic library that is made using bacteriophages is stored as a collection of phage clones. A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert. This minimizes the number of clones needed.

or Recombinant phage DNA Foreign genome cut up with restriction enzyme Large plasmid Large insert with many genes BAC clone Bacterial clones Recombinant plasmids Phage clones (a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones

Storing Cloned Genes in DNA Libraries A complementary DNA (cdna) library is made by cloning DNA made in vitro by reverse transcription of all the mrna produced by a particular cell. To do this, mrna is extracted from cells and reverse transcribed to produce DNA that is complementary to the mrna. A cdna library represents only part of the genome only the subset of genes transcribed into mrna in the original cells.

DNA in nucleus mrnas in cytoplasm mrna Reverse transcriptase Poly-A tail Degraded mrna DNA strand Primer DNA polymerase cdna

Screening a Library for Clones Carrying a Gene of Interest Once clones have be cultured that contain the foreign DNA, clones carrying the gene of interest must be identified. A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene. The nucleic acid probe is usually labelled somehow (radioactivity, luminescence, etc.) so that its presence can be identified. This process is called nucleic acid hybridization.

Screening a Library for Clones Carrying a Gene of Interest A probe can be synthesized that is complementary to the gene of interest. For example, if the desired gene is 5 GGCT AACT T AGC Then we would synthesize this probe 3 CCGAT T GAAT CG The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest through complementary base pairing. Once identified, the clone carrying the gene of interest can be cultured to amplify the gene of interest. 3 5

TECHNIQUE Multiwell plates holding library clones Radioactively labeled probe molecules Probe DNA Gene of interest Single-stranded DNA from cell Film Nylon membrane Nylon Location of membrane DNA with the complementary sequence

Bacterial Expression Systems After a gene has been cloned, its protein product can be produced in larger amounts for research. Cloned genes can be expressed as protein in either bacterial or eukaryotic cells. However, due to differences between bacterial and eukaryotic gene expression, several obstacles must be overcome. To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter upstream of the restriction site for insertion of the eukaryotic gene. To overcome the lack of RNA-splicing, a cdna form of a gene can be used.

Eukaryotic Cloning and Expression Systems The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems. YACs behave normally in mitosis and can carry more DNA than a plasmid. Eukaryotic hosts can provide the post-translational modifications that many proteins require. One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes. Alternatively, scientists can inject DNA into cells using microscopically thin needles. Once inside the cell, the DNA can be incorporated into the cell s DNA by genetic recombination.

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) While DNA cloning in cells is effective for cloning large quantities of a gene, other methods can also be used to amplify DNA. The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA without the use of a cell. A three-step cycle heating, cooling, and replication brings about a chain reaction that produces an exponentially growing population of identical DNA molecules. The DNA polymerase used is stable at high temperatures.

TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 1 Denaturation 5 3 2 Annealing 3 5 Cycle 1 yields 2 molecules 3 Extension Primers New nucleotides

Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

Concept 20.2 DNA technology allows us to study the sequence, expression, and function of a gene DNA cloning allows researchers to compare genes and alleles between individuals Locate gene expression in a body Determine the role of a gene in an organism Several techniques can be used to analyze the samples obtained from DNA cloning.

Gel Electrophoresis 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 (nucleic acids carry a negative charge on their phosphate groups). Molecules are sorted into bands by their size. Larger molecules generally migrate slower through the gel than smaller molecules. A ladder containing fragments of known sizes can be used to estimate sizes of the fragments.

TECHNIQUE Mixture of DNA molecules of different sizes Power source Cathode Anode + 1 Gel Longer molecules Power source + 2 Shorter molecules

RESULTS

Restriction Fragment Analysis 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 which can be isolated and recovered from the gel. However, larger DNA molecules will result in a smear of different sized fragments of DNA, rather than distinct bands.

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

Southern Blotting 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 the gel on a nitrocellulose membrane. This is useful when too many bands are produced and the sequence of interest cannot be resolved from the other bands in a gel.

TECHNIQUE DNA + restriction enzyme Restriction fragments I II III Nitrocellulose membrane (blot) Heavy weight Gel Sponge I Normal -globin allele II Sickle-cell allele III Heterozygote Alkaline solution Paper towels 1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting) Radioactively labeled probe for -globin gene I II III Probe base-pairs with fragments I II III Nitrocellulose blot Fragment from sickle-cell -globin allele Fragment from normal -globin allele 4 Hybridization with radioactive probe 5 Probe detection Film over blot

DNA Sequencing Once a fragment of DNA is cloned, its sequence can be determined through the dideoxyribonucleotide chain termination method, developed by Frederick Sanger. Modified nucleotides called dideoxyribonucleotides (ddntp) attach to synthesized DNA strands of different lengths. Each type of ddntp is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment. The DNA sequence can be read from the resulting spectrogram.

TECHNIQUE DNA (template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) DNA polymerase datp dctp dttp dgtp ddatp ddctp ddttp ddgtp

TECHNIQUE DNA (template strand) Labeled strands Shortest Longest

TECHNIQUE Direction of movement of strands Longest labeled strand Detector RESULTS Laser Last base of longest labeled strand Shortest labeled strand Last base of shortest labeled strand

Studying the Expression of Single Genes Nucleic acid probes can hybridize with mrnas transcribed from a gene These probes can be used to identify where or when a gene is transcribed in an organism For example, changes in the expression of a gene during embryonic development can be tested using: Northern blotting Reverse transcriptase-polymerase chain reaction Both methods are used to compare mrna from different developmental stages

Northern Blotting Northern blotting combines gel electrophoresis of mrna (instead of DNA like in the Southern blot) followed by hybridization with a probe on a membrane. Detection of a hybridized probe would indicate the presence of mrna with that particular sequence in that sample. Identification of mrna at a particular developmental stage suggests that possibly protein functions at that stage as well.

RT-PCR Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive than a Northern blot. 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 and quantified in different samples.

TECHNIQUE 1 cdna synthesis mrnas 2 PCR amplification Primers cdnas 3 Gel electrophoresis -globin gene RESULTS Embryonic stages 1 2 3 4 5 6

In situ hybridization In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mrnas in place in the intact organism 50 µm

Studying the Expression of Interacting Groups of Genes Now that entire genomes of various organisms have been sequenced, it is possible to look at expression of large groups of genes to determine which genes are transcribed in different situations. Groups of genes that are expressed coordinately can also be found. 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

TECHNIQUE 1 Isolate mrna. Tissue sample 2 Make cdna by reverse transcription, using fluorescently labeled nucleotides. mrna molecules 3 Apply the cdna mixture to a microarray, a different gene in each spot. The cdna hybridizes with any complementary DNA on the microarray. Labeled cdna molecules (single strands) DNA fragments representing specific genes DNA microarray 4 Rinse off excess cdna; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. DNA microarray with 2,400 human genes

Determining Gene Function One way to determine function is to disable the gene and observe the consequences. Using in vitro mutagenesis, specific mutations are introduced into a cloned gene, to knock out or disable the gene. 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) sirnas can be used to break down or block the gene s mrna