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Recombinant DNA Technology

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1 Recombinant DNA Technology

2 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectors © 2012 Pearson Education, Inc.

3 Section 20.1 Recombinant DNA refers to the joining of DNA molecules, usually from different biological sources, that are not found together in nature © 2012 Pearson Education, Inc.

4 The basic procedure for producing recombinant DNA involves
Section 20.1 The basic procedure for producing recombinant DNA involves generating specific DNA fragments using restriction enzymes joining these fragments with a vector transferring the recombinant DNA molecule to a host cell to produce many copies that can be recovered from the host cell © 2012 Pearson Education, Inc.

5 Section 20.1 The recovered copies of a recombinant DNA molecule are referred to as clones and can be used to study the structure and orientation of the DNA Recombinant DNA technology is used to isolate, replicate, and analyze genes © 2012 Pearson Education, Inc.

6 Figure 20-1 Common restriction enzymes, with their restriction sites, DNA cutting patterns, and sources. Arrows indicate the location in the DNA cut by each enzyme. Figure 20.1 © 2012 Pearson Education, Inc.

7 Figure 20-2 DNA from different sources is cleaved with EcoRI and mixed to allow annealing. The enzyme DNA ligase forms phosphodiester bonds between these fragments to create an intact recombinant DNA molecule. Figure 20.2 © 2012 Pearson Education, Inc.

8 Section 20.1 Vectors are carrier DNA molecules that can replicate cloned DNA fragments in a host cell Vectors must be able to replicate independently and should have several restriction enzyme sites to allow insertion of a DNA fragment Vectors should carry a selectable gene marker to distinguish host cells that have taken them up from those that have not © 2012 Pearson Education, Inc.

9 Section 20.1 A plasmid is an extrachromosomal double-stranded DNA molecule that replicates independently from the chromosomes within bacterial cells (Figure 20.3a) © 2012 Pearson Education, Inc.

10 Figure 20-3 A color-enhanced electron micrograph of circular plasmid molecules isolated from E. coli. Genetically engineered plasmids are used as vectors for cloning DNA. (b) A diagram of a typical DNA cloning plasmid. Figure 20.3 © 2012 Pearson Education, Inc.

11 Figure 20.4 © 2012 Pearson Education, Inc.

12 Figure 20-5 In blue-white selection procedures, DNA inserted into multiple cloning site of a plasmid disrupts the lacZ gene so that bacteria containing recombinant DNA are unable to metabolize X-gal, resulting in white colonies that allow direct identification of bacterial colonies carrying cloned DNA inserts. Photo of a Petri dish showing the growth of bacterial cells after uptake of recombinant plasmids. Cells in blue colonies contain vectors without cloned DNA inserts, whereas cells in white colonies contain vectors carrying Dna inserts. Figure 20.5 © 2012 Pearson Education, Inc.

13 Vectors Carry DNA Molecules to Be Cloned
Lambda () Phage Vectors © 2012 Pearson Education, Inc.

14  phage as a vector. DNA is extracted from the phage, the central gene cluster is removed, and the DNA to be cloned is ligated into the arms of the  chromosome. The recombinant chromosome is then packaged into phage proteins to form a recombinant virus. © 2012 Pearson Education, Inc.

15 Vectors Carry DNA Molecules to Be Cloned
Cosmid Vectors © 2012 Pearson Education, Inc.

16 The cosmid pJB8 contains a bacterial origin of replication (ori), a single cos sequence (cos), an ampicillin resistance gene (amp, for selection of colonies that have taken up the cosmid), and a region containing four sites for cloning (BamHI, EcoRI, ClaI, and HindIII). Because the vector is small (5.4 kb long), it can accept foreign DNA segments between 33 and 46 kb in length. The cos sequence allows cosmids carrying large inserts to be packaged into lambda viral coat proteins as though they were viral chromosomes. The viral coats carrying the cosmid can be used to infect a suitable bacterial host, and the vector, carrying a DNA insert, will be transferred into the host cell. Once inside, the ori sequence allows the cosmid to replicate as a bacterial plasmid. © 2012 Pearson Education, Inc.

17 Vectors Carry DNA Molecules to Be Cloned
Bacterial Artificial Chromosomes © 2012 Pearson Education, Inc.

18 A bacterial artificial chromosome (BAC)
A bacterial artificial chromosome (BAC). The polylinker carries a number of unique sites for the insertion of foreign DNA. The arrows labeled T7 and Sp6 are promoter regions that allow expression of genes cloned between these regions. © 2012 Pearson Education, Inc.

19 Vectors Carry DNA Molecules to Be Cloned
Expression Vectors © 2012 Pearson Education, Inc.

20 A pET expression vector
A pET expression vector. This system uses a genetically engineered host cell. The host cell carries the viral T7 RNA polymerase gene under the control of a lac promoter and operator, making it inducible by the lactose analog IPTG. For expression of a target gene, pET vectors carrying the target gene are inserted into host cells. Growth of the host on IPTG derepresses the T7 RNA polymerase gene and the target gene in the pET vector (which is under lac O control), leading to expression of the target gene. This system combines a strong promoter with tight regulation, and expression only occurs in the presence of IPTG. © 2012 Pearson Education, Inc.

21 DNA Was First Cloned in Prokaryotic Host Cells
© 2012 Pearson Education, Inc.

22 Yeast Cells Are Used as Eukaryotic Hosts for Cloning
© 2012 Pearson Education, Inc.

23 Table 13-1 Recombinant Proteins Synthesized in Yeast Cells
© 2012 Pearson Education, Inc.

24 The yeast artificial chromosome pYAC3 contains telomere sequences (TEL), a centromere (CEN4) derived from yeast chromosome 4, and an origin of replication (ori). These elements give the cloning vector the properties of a chromosome. TRP1 and URA3 are yeast genes that are selectable markers for the left and right arms of the chromosome. Within the SUP4 gene is a restriction enzyme recognition sequence for the enzyme SnaB1. Two BamH1 recognition sequences flank a spacer segment. Cleavage with SnaB1 and BamH1 breaks the artificial chromosome into two arms. The DNA to be cloned is treated with SnaB1 producing a collection of fragments. The arms and fragments are ligated together, and the artificial chromosome is inserted into yeast host cells. Because yeast chromosomes are large, the artificial chromosome accepts inserts in the million base-pair range. © 2012 Pearson Education, Inc.

25 Plant and Animal Cells Can Be Used As Host Cells For Cloning
Plant Cell Hosts © 2012 Pearson Education, Inc.

26 From Agrobacterium tumifaciens
A Ti plasmid designed for cloning in plants. Segments of T-DNA, including those necessary for integration, are combined with bacterial segments that incorporate cloning sites and antibiotic resistance genes (kanR and tetR). The vector also contains an origin of replication (ori), as well as a lambda cos sequence that permits recovery of cloned inserts from the host plant cell. From Agrobacterium tumifaciens © 2012 Pearson Education, Inc.

27 Section 20.1 A variety of different human cell types can be grown in culture and used to express genes and proteins These lines can be subjected to various approaches for gene or protein functional analysis, including drug testing for effectiveness at blocking or influencing a particular recombinant protein being expressed, especially if the cell lines are of a human disease condition such as cancer © 2012 Pearson Education, Inc.

28 Cloned DNA can be transferred into mammals by direct injection into the oocytes.
© 2012 Pearson Education, Inc.

29 20.2 DNA Libraries Are Collections of Cloned Sequences
© 2012 Pearson Education, Inc.

30 Section 20.2 DNA libraries represent a collection of cloned DNA samples derived from a single source that could be a particular tissue type, cell type, or single individual A genomic library contains at least one copy of all the sequences in the genome of interest Genomic libraries are constructed by cutting genomic DNA with a restriction enzyme and ligating the fragments into vectors, which are chosen depending on the size of the genome © 2012 Pearson Education, Inc.

31 Section 20.2 Complementary DNA (cDNA) libraries contains complementary DNA copies made from the mRNAs present in a cell population and represents the genes that are transcriptionally active at the time the cells were collected for mRNA isolation © 2012 Pearson Education, Inc.

32 Figure 20-6 Producing cDNA from mRNA
Figure 20-6 Producing cDNA from mRNA. Because many eukaryotic mRNAs have a poly-A tail of variable length at one end, a short oligo-dT annealed to this tail serves as a primer for the enzyme reverse transcriptase. Reverse transcriptase uses the mRNA as a template to synthesize a complementary DNA strand (cDNA) and forms an mRNA/cDNA double-stranded duplex. The mRNA is digested with the enzyme RNAse H, producing gaps in the RNA strand. The 3’ ends of the remaining RNA serves as a primer for DNA polymerase I, which synthesizes a second DNA strand. The result is a double-stranded cDNA molecule that can be cloned into a suitable vector, or used directly as a probe for library screening. Figure 20.6 © 2012 Pearson Education, Inc.

33 Figure 20-7 Screening a library constructed using a plasmid vector to recover a specific gene. The library, present in bacteria on Petri plates, is overlaid with a DNA-binding membrane, and colonies are transferred to the membrane. Colonies on the membrane are lysed, and the DNA is denatured to single strands. The membrane is placed in a hybridization bag along with buffer and a labeled single-stranded DNA probe. During incubation, the probe forms a double-stranded hybrid with any complementary sequences on the membrane. The membrane is removed from the bag and washed to remove excess probe. Hybrids are detected by placing a piece of X-ray film over the membrane and exposing it for a short time. The film is developed, and hybridization events are visualized as spots on the film. Colonies containing the insert that hybridized to the probe are identified from the orientation of the spots. Cells are picked from this colony for growth and further analysis. Figure 20.7 © 2012 Pearson Education, Inc.

34 20.3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA
© 2012 Pearson Education, Inc.

35 Figure 20-8 In the polymerase chain reaction (PCR), the target DNA is denatured into single strands; each strand is then annealed to short, complementary primers. DNA polymerase extends the primers in the 5' to 3' direction, using the single-stranded DNA as a template. The result after one round of replication is a doubling of DNA molecules to create two newly synthesized double-stranded DNA molecules. Repeated cycles of PCR can quickly amplify the original DNA sequence more than a millionfold. Note: shown here is a relatively short sequence of DNA being amplified. Typically much longer segments of DNA are used for PCR and the primers bind somewhere within the DNA molecule and not so close to the end of the actual molecule. Figure 20.8 © 2012 Pearson Education, Inc.

36 Section 20.3 Reverse transcription PCR (RT-PCR) is used to study gene expression by studying mRNA production by cells or tissues Quantitative real-time PCR (qPCR) or real-time PCR allows researchers to quantify amplification reactions as they occur in ‘real time’ (Figure 20.9) The procedure uses an SYBR green dye and TaqMan probes, which contain two dyes © 2012 Pearson Education, Inc.

37 Figure 20.9 © 2012 Pearson Education, Inc.

38 20.4 Molecular Techniques for Analyzing DNA
© 2012 Pearson Education, Inc.

39 Section 20.4 A restriction map establishes the number and order of restriction sites and the distance between restriction sites on a cloned DNA segment It provides information about the length of the cloned insert and the location of restriction sites within the clone © 2012 Pearson Education, Inc.

40 Figure An agarose gel containing separated DNA fragments stained with the DNA-binding dye (ethidium bromide) and visualized under ultraviolet light. Smaller fragments migrate faster and farther than do larger fragments, resulting in the distribution shown. Molecular techniques involving agarose gel electrophoresis are routinely used in a wide range of applications. Figure 20.10 © 2012 Pearson Education, Inc.

41 Constructing a restriction map. Samples of the 7
Constructing a restriction map. Samples of the 7.0-kb DNA fragments are digested with restriction enzymes: One sample is digested with HindIII, one with SalI, and one with both HindIII and SalI. The resulting fragments are separated by gel electrophoresis. The separated fragments are measured by comparing them with molecular-weight standards in an adjacent lane. Cutting the DNA with HindIII generates two fragments: 0.8 kb and 6.2 kb. Cutting with SalI produces two fragments: 1.2 kb and 5.8 kb. Models are constructed to predict the fragment sizes generated by cutting with HindIII and with SalI. Model 1 predicts that 0.4-, 0.8-, and 5.8-kb fragments will result from cutting with both enzymes. Model 2 predicts that 0.8-, 1.2-, and 5.0-kb fragments will result. Comparing the predicted fragments with those observed on the gel indicates that model 1 is the correct restriction map. © 2012 Pearson Education, Inc.

42 Figure In the Southern blotting technique, samples of the DNA to be probed are cut with restriction enzymes and the fragments are separated by gel electrophoresis. The pattern of fragments is visualized and photographed under ultraviolet illumination. Then the gel is placed on a sponge wick that is in contact with a buffer solution and covered with a DNA-binding membrane. layers of paper towels or blotting paper are placed on top of the membrane and held in place with a weight. Capillary action draws the buffer through the gel, transferring the pattern of DNA fragments from the gel to the membrane. The DNA fragments on the membrane are then denatured into single strands and hybridized with a labeled DNA probe. The membrane is washed to remove excess probe and overlaid with a piece of X-ray film for autoradiography. The hybridized fragments show up as bands on the X-ray film. Figure 20.11 © 2012 Pearson Education, Inc.

43 © 2012 Pearson Education, Inc.

44 © 2012 Pearson Education, Inc.

45 Section 20.4 Northern blot analysis is used to determine whether a gene is actively being expressed in a given cell or tissue Used to study patterns of gene expression in embryonic tissues, cancer, and genetic disorders © 2012 Pearson Education, Inc.

46 Figure 20.14 © 2012 Pearson Education, Inc.

47 20.5 DNA Sequencing Is the Ultimate Way to Characterize DNA Structure at the Molecular Level
© 2012 Pearson Education, Inc.

48 Figure 20.15 © 2012 Pearson Education, Inc.

49 DNA sequencing using the chain termination method
DNA sequencing using the chain termination method. (1) A primer is annealed to a sequence adjacent to the DNA being sequenced (usually at the insertion site of a cloning vector). (2) A reaction mixture is added to the primer–template combination. This includes DNA polymerase, the four dNTPs (one of which is radioactively labeled) and a small amount of one dideoxynucleotide. Four tubes are used, each containing a different dideoxynucleotide (ddATP, ddCTP, etc.). (3) During primer extension, the polymerase occasionally inserts a ddNTP instead of a dNTP, terminating the synthesis of the chain, because the ddNTP does not have the -OH group needed to attach the next nucleotide. In the figure, ddATP and the A inserted from this dideoxynucleotide are indicated with an asterisk. Over the course of the reaction, all possible termination sites will have a ddNTP inserted. (4) The newly synthesized strands are removed from the template, and the mixture is placed on a gel. DNA fragments from the reaction tube containing ddATP and terminating with A are loaded in the A lane, those ending in C are loaded in the C lane, and so forth. © 2012 Pearson Education, Inc.

50 Figure 19-27 Copyright © 2006 Pearson Prentice Hall, Inc.
Figure DNA sequencing gel showing the separation of newly synthesized fragments in the four sequencing reactions (one per lane). To obtain the base sequence of the DNA fragment, the gel is read from the bottom, beginning with the lowest band in any lane, then the next lowest, then the next, and so on. For example, the sequence of the DNA on this gel begins with -TT at the very bottom of the gel, and proceeds upwards as -TTCGTGAAGAA and so forth. 5’-TTCGTGAA…etc A A G T C G T T Figure Copyright © 2006 Pearson Prentice Hall, Inc. © 2012 Pearson Education, Inc.

51 Figure Computer-automated DNA sequencing using the chain-termination (Sanger) method. (1) A primer is annealed to a sequence adjacent to the DNA being sequenced (usually near the multiple cloning site of a cloning vector). (2) A reaction mixture is added to the primer–template combination. This includes DNA polymerase, the four dNTPs, and small molar amounts of dideoxy-nucleotides (ddNTPs) labeled with fluorescent dyes. All four ddNTPs are added to the same tube, and during primer extension, all possible lengths of chains are produced. During primer extension, the polymerase occasionally (randomly) inserts a ddNTP instead of a dNTP, terminating the synthesis of the chain because the ddNTP does not have the OH group needed to attach the next nucleotide. Over the course of the reaction, all possible termination sites will have a ddNTP inserted. The products of the reaction are added to a single lane on a capillary gel, and the bands are read by a detector and imaging system. This process is now automated, and robotic machines, such as those used in the Human Genome Project, sequence several hundred thousand nucleotides in a 24-hour period and then store and analyze the data automatically. The sequence is obtained by extension of the primer and is read from the newly synthesized strand, not the template strand. Thus, the sequence obtained begins with 5´-ctagacatg-3´. Figure 20.16 © 2012 Pearson Education, Inc.

52 Figure Output of a computer-automated DNA sequencing chromatograph or electropherogram. Each peak represents the correct nucleotide in the sequence. The sequence extending from the primer (which is not shown here) starts at the upper left of the diagram and extends to the right. The bases labeled as n are ambiguous and cannot be identified with certainty. These ambiguous base readings are more likely to occur near the primer because the quality of sequence determination deteriorates the closer the sequence is to the primer. The separated bases are read in order along the axis from left to right. Thus, this sequence begins as 5´-TGNNANACTGACNCAC. Numbers below the bases indicate length of the sequence in base pairs. Figure 20.17 © 2012 Pearson Education, Inc.

53 Figure 20.18 © 2012 Pearson Education, Inc.

54 Figure 20.19 © 2012 Pearson Education, Inc.

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