DNA Sequencing 20.3 PDF

20.3 DNA Sequencing 545 Using Standards to Determine the Initial Concentration of the DNA sequence. For this reason, analyzing DNA sequences is Template DNA. To determine the amount of starting template a powe...

20.3 DNA Sequencing 545 Using Standards to Determine the Initial Concentration of the DNA sequence. For this reason, analyzing DNA sequences is Template DNA. To determine the amount of starting template a powerful approach for understanding genetics. A technique DNA, the sample of interest that has an unknown amount of starting called DNA sequencing enables researchers to determine the base template DNA is compared with some type of standard. qPCR in- sequence of DNA found in genes and other chromosomal regions. volves the coamplification of two templates: the sample of interest It is one of the most important tools for exploring genetics at the and the standard. The two types of PCR products can be monitored molecular level. Molecular geneticists often want to determine simultaneously using molecules that fluoresce in different colors. DNA base sequences as a first step toward understanding the func- What types of standards are commonly used? One possibility tion and expression of genes. For example, the investigation of is a standard of known concentration that is added to the PCR mix- genetic sequences has been vital to our understanding of promot- ture. For example, plasmid DNA carrying a specific gene can be ers, regulatory elements, and the genetic code itself. Furthermore, added to the PCR mixture in known amounts, and the amplification an examination of sequences has facilitated our understanding of of this plasmid-coded gene provides a standard. By comparing the origins of replication, centromeres, telomeres, and transposable Ct values for the standard and the unknown sample, researchers can elements. determine the concentration of the unknown sample. This is shown During the 1970s, two methods for DNA sequencing were schematically in Figure 20.10c, but is actually accomplished with devised. One method, developed by Allan Maxam and Walter computer software. Alternatively, researchers may use an internal Gilbert, involved the base-specific chemical cleavage of DNA. standard in which another gene that is already present in the sample Another method, developed by Frederick Sanger and colleagues, is also amplified. For example, a gene that codes the cytoskeletal is known as dideoxy sequencing. Gilbert and Sanger won the protein called actin may be used. This relative quantitation method 1980 Nobel Prize in Chemistry for their contributions to DNA is somewhat simpler. The amount of unknown template DNA of sequencing. Because it became the more popular method of DNA interest is expressed relative to the internal standard. sequencing, we will consider the dideoxy method here. In addi- tion, Chapter 22 considers some newer methods of sequencing DNA that are not based on the Sanger dideoxy method. These 20.2 COMPREHENSION QUESTIONS newer methods are commonly used in projects aimed at determin- ing the DNA sequence of an entire genome. 1. In one PCR cycle, the correct order of steps is The dideoxy method of DNA sequencing is based on our a. primer annealing, primer extension, denaturation. knowledge of DNA replication but includes a clever twist. As b. primer annealing, denaturation, primer extension. described in Chapter 11, DNA polymerase connects adjacent c. denaturation, primer annealing, primer extension. deoxyribonucleotides by catalyzing formation of a covalent bond between the 5′ phosphate on one nucleotide and the d. denaturation, primer extension, primer annealing. 3′─OH group on the previous nucleotide (refer back to 2. In reverse transcriptase PCR, the starting biological material is Figure 11.16). However, chemists can synthesize deoxyribonu- a. chromosomal DNA. cleotides that are missing the ─OH group at the 3′ position b. mRNA. (Figure 20.11). These synthetic nucleotides are called dideoxy- c. proteins. ribonucleotides (ddNTPs). [Note: The prefix dideoxy- indi- d. all of the above. cates that two (di) oxygens (oxy) have been removed (de) from 3. During qPCR, the synthesis of PCR products is analyzed the nucleotide’s sugar; in comparison, ribose has ─OH groups a. at the very end of the reaction by gel electrophoresis. at both the 2′ and 3′ positions.] Sanger reasoned that if a dide- oxyribonucleotide is added to a growing DNA strand, the strand b. at the very end of the reaction by fluorescence that is emit- can no longer grow because the dideoxyribonucleotide is miss- ted within the thermocycler. ing the 3′ ─OH group. The incorporation of a dideoxyribonu- c. during the PCR cycles by gel electrophoresis. cleotide into a growing DNA strand is therefore referred to as d. during the PCR cycles by fluorescence that is emitted within chain termination. the thermocycler. O O O Adenine 20.3 DNA SEQUENCING O– P O P O P O CH2 5′ O Learning Outcome: O– O– O– 1′ 4′ H H H H 1. Outline the steps in automated DNA sequencing via the 3′ 2′ dideoxy method. H H 2′,3′-Dideoxyadenosine triphosphate (ddA) As we have seen throughout this text, our knowledge of genetics F I G URE 2 0. 1 1 The structure of a dideoxyribonucleotide. can be largely attributed to an understanding of DNA structure and Note that the 3′ group is a hydrogen rather than an ─OH group. For this function. The feature that underlies all aspects of inherited traits is reason, another nucleotide cannot be attached at the 3′ position. bro50795_ch20_530-560.indd 545 17/06/23 11:43 AM  546 C H A P T E R 2 0 :: MOLECULAR TECHNOLOGIES To detect the incorporation of dideoxyribonucleotides during following: ddA is green, ddT is red, ddG is yellow, and ddC is blue. DNA replication, the newly made DNA strands must be labeled in This newer method is called automated DNA sequencing because some way. When dideoxy sequencing was first invented, researchers the sequence of colored nucleotides is read by a machine. labeled the nucleotides with radioisotopes, which allowed the newly For automated DNA sequencing, the segment of DNA to be made strands to be detected via autoradiography. This traditional sequenced is obtained in large amounts. This can be accomplished DNA sequencing method was modified by labelling each type of using gene cloning, which was described earlier in this chapter. In dideoxyribonucleotide with a different-colored fluorescent dye. For the example in Figure 20.12, the segment of DNA to be sequenced, example, a common way to fluorescently label ddNTPs is the which we will call the target DNA, was cloned into a vector at a G C A T T C FIGURE 20.12 The pro- G C Sequence to tocol for DNA sequencing by the T be analyzed dideoxy method. (a) The method T G G Primer (target DNA) begins with a recombinant vector A C Annealing into which the target DNA has been inserted. For the primer to site bind, the recombinant vector must be denatured into single- 5′ stranded DNA at the beginning of the experiment. Only the strand needed for DNA sequencing is shown here. This diagram schemat- Recombinant vector ically depicts a series of bands on a gel; the four colors of the bands occur because each type of dideoxyribonucleotide is labeled with a different-colored fluorescent dye. As each band passes a la- ser, the fluorescent dye is excited by the laser beam, and the fluo- Many copies of the recombinant vector and primer rescence emission is recorded by a machine with a fluorescence are mixed together. Normal dNTPs are added detector. The detector reads the level of fluorescence at four wave- at a high concentration, and fluorescently labeled ddNTPs are added at a low concentration. lengths corresponding to the colors of the four dyes. (b) As shown DNA polymerase is then added. in the printout, the peaks of fluorescence correspond to the DNA sequence that is complementary to the target DNA. Note that ddG CACCGTAAGGACTddG CACCGTAAGGACddT is usually labeled with a yellow dye, but black ink is used instead of CACCGTAAGGAddC yellow on the printout shown in Figure 20.12b for ease of reading. CACCGTAAGGddA CONCEPT CHECK: What are two key ways that the DNA segments CACCGTAAGddG forming the bands on the gel differ from each other? CACCGTAAddG CACCGTAddA Nucleotides added to primer CACCGTddA CACCGddT CACCddG CACddC CAddC CddA ddC Separate newly made strands by gel electrophoresis. Sequence deduced from gel G T C A C A C C G T A A G G A C T G G G A A T G C C A Laser Fluorescence C beam detector (a) Automated DNA sequencing (b) Output from automated sequencing bro50795_ch20_530-560.indd 546 17/06/23 11:43 AM  20.4 Gene Editing via CRISPR-Cas Technology 547 defined location. The target DNA was inserted next to a site in the Theoretically, it is possible to read this base sequence di- vector where a primer will bind, which is called the annealing site. rectly from the gel. From a practical perspective, however, it is The aim of the experiment is to determine the base sequence of the faster and more efficient to automate the procedure using a ma- target DNA next to the annealing site. In the experiment shown in chine with a laser and a fluorescence detector. As the gel is run- Figure 20.12, the recombinant vector DNA has been previously ning, each band passes the laser and the laser beam excites the denatured into single strands, usually via heat treatment. Only the fluorescent dye. The fluorescence detector records the amount of strand needed for DNA sequencing is shown here. fluorescence emission from the excited dye. The detector reads the Let’s now consider the steps that are involved in DNA level of fluorescence at four wavelengths, corresponding to the sequencing by the dideoxy method (Figure 20.12a). four different colors of the dyes. An example of the printout from the fluorescence detector is shown in Figure 20.12b. As can be 1. A sample containing many copies of the single-stranded seen there, the peaks of fluorescence correspond to the DNA se- recombinant vector is mixed with many primers that will quence that is complementary to the target DNA. Though im- bind to the annealing site. The primer binds to the DNA provements in automated sequencing continue to be made, a because it is complementary to the annealing site. typical sequencing run can determine a DNA sequence that is ap- 2. All four types of deoxyribonucleotides are added at a high proximately 700–900 bases long, or perhaps even longer. concentration, and all four dideoxyribonucleotides (ddA, ddT, ddG, and ddC), which are fluorescently labeled, are added at a low concentration. 3. DNA polymerase is then added, which causes the synthesis 20.3 COMPREHENSION QUESTION of strands that are complementary to the target DNA se- 1. When a dideoxyribonucleotide is incorporated into a growing quence. DNA synthesis continues until a dideoxyribonucle- DNA strand, otide is incorporated into a growing strand. For example, a. the strand elongates faster. chain termination can occasionally occur at the sixth or b. the strand cannot elongate. thirteenth position of the newly synthesized DNA strand if a ddT becomes incorporated at either of these sites. Note c. the strand becomes more susceptible to DNase I cleavage. that the base A (adenine), the base that is complementary d. none of the above events occurs. to T (thymine), is found at the sixth and thirteenth position in the target DNA. Therefore, we expect to obtain DNA strands that terminate at the sixth or thirteenth positions and have a ddT at their ends. Because these DNA strands 20.4 GENE EDITING VIA contain a ddT, they are fluorescently labeled in red. CRISPR-CAS TECHNOLOGY Alternatively, ddA causes chain termination at the second, seventh, eighth, or eleventh position because the comple- Learning Outcomes: mentary base T base is found at the corresponding position 1. Describe the method of CRISPR-Cas technology, and give in the target strand. Strands that are terminated with ddA examples of its uses. are fluorescently labeled in green. 2. Explain how dead Cas9 can be used to increase the expres- 4. After the samples have been incubated for several minutes, sion of genes. mixtures of DNA strands of different lengths are made, de- 3. Discuss some ethical considerations concerning human pending on the number of nucleotides attached to the gene editing. primer. These DNA strands are separated according to their lengths by running them on a slab gel or more commonly To understand how the genetic material functions, researchers often by running them through a gel-filled capillary tube. The analyze mutations that alter normal DNA sequences, thereby affect- shorter strands move to the bottom of the gel more quickly ing the expression of genes and the outcome of traits. For example, than the longer strands. geneticists have discovered that many inherited human diseases, As mentioned, each of the four types of bases in a dideoxynucleo- such as sickle cell disease and hemophilia, involve mutations within tide is labeled with a different color. Because the incorporation of specific genes. These mutations provide insight into the function of a dideoxynucleotide stops the further growth of a DNA strand, the genes in unaffected individuals. Hemophilia, for example, is only the last nucleotide in a strand is labeled. For example, if a ddT caused by mutations in genes that code blood-clotting factors. was incorporated at the end of a DNA strand, the strand would be Because the analysis of mutations can provide important red, but if a ddC was incorporated, the strand would be blue. The information about normal genetic processes, researchers often DNA sequence that is complementary to the target DNA is de- wish to produce mutant organisms. As discussed in Chapter 19, duced by determining the sequence of colors in a series of DNA mutations can arise spontaneously or can be induced by environ- strands that differ in their lengths. Reading the base sequence, mental agents. Mendel’s pea plants are a classic example of allelic from bottom to top, is much like climbing a ladder of colored strains with different phenotypes that arose from spontaneous mu- bands. For this reason, the series of bands obtained by this method tations. In addition, experimental organisms can be treated with is referred to as a sequencing ladder (see Figure 20.12a). mutagens that increase the rate of mutations. bro50795_ch20_530-560.indd 547 17/06/23 11:43 AM