The Sanger method for DNA sequencing was first described almost 30 years ago (Sanger et al. 1977). This Nobel Prize winning technique is still employed in modern-day DNA sequencing. The process involves the polymerase incorporation of dideoxyribonucleotide triphosphates (ddNTPs) as chain terminators
DNA template 5'-
AT AT AT AT AT AT
TO TA O TAC
Extension produces a series of ddNTP terminated products each one base different in length
TACT O TACTA C TACTAAO TACTAAGO
Each ddNTP is labeled with a different color fluorescent dye
ATTTACTAAGG 270 2
Sequence is read by noting peak color in electropherogram (possessing single base resolution)
followed by a separation step capable of single nucleotide resolution. There is no hydroxyl group at the 3'-end of the DNA nucleotide (see Figure 2.1) with a ddNTP and therefore chain growth terminates when the polymerase incorporates a ddNTP into the synthesized strand. Extendable dNTPs and ddNTP terminators are both present in the reaction mix so that some portions of the DNA molecules are extended. At the end of sequencing reaction a series of molecules are present that differ by one base from one another.
Figure 10.5 illustrates the Sanger sequencing process. Each DNA strand is sequenced in separate reactions with a single primer. Often either the forward or reverse PCR primers are used for this purpose. Four different colored fluorescent dyes are attached to the four different ddNTPs. Thus, ddTTP (thymine) is labeled with a red dye, ddCTP (cytosine) is labeled with a blue dye, ddATP (adenine) is labeled with a green dye, and ddGTP (guanine) is labeled with a yellow dye although it is typically displayed in black for easier visualization. These are similar dyes as will be described in Chapter 13 for STR detection. Fluorescent dye labels have simplified DNA sequencing as have the widespread use of automated detection systems and capillary electrophoresis. The Human Genome Project was completed with these sequencing technologies.
Validation of various DNA sequencing chemistries has progressed over the past decade from a simple Taq polymerase, which often had high backgrounds and poor incorporation rates for many nucleotide combinations to the well-balanced Big Dye chemistries used today. Signal-to-noise ratios have improved
DNA sequencing process with fluorescent ddNTPs. A primer that has been designed to recognize a specific region of a DNA template anneals and is extended with a polymerase. Because a mixture of dNTPs and ddNTPs exist for each of the four possible nucleotides, some of the extension products are halted by incorporation of a ddNTP while other molecules continue to be extended. Each ddNTP is labeled with a different dye that enables each extension product to be distinguished by color. A size-based separation of the extension products permits the DNA sequence to be read provided that sufficient resolution is present to clearly see each base.
with brighter dyes (Lee et al. 1997), which in turn now permits obtaining results from less material. As little as 1 ng of mtDNA PCR product can now be used for each DNA sequencing reaction (Stewart et al. 2003).
DNA sequencing of mtDNA is usually performed with the following steps: (1) PCR amplification of the entire control region or a portion of it with various primer sets as will be explained below; (2) removal of remaining dNTPs and primers from PCR through spin filtration using a Microcon 100 filter or enzymatic digestion with shrimp alkaline phosphatase and exonuclease I (Dugan et al. 2002); (3) determination of PCR product quantity (Wilson et al. 1995a, 1995b); (4) performance of DNA sequencing reaction to incorporate fluorescent ddNTPs as described above with each reaction containing a different primer to dictate which strand is sequenced; (5) removal of unincorporated fluorescent dye terminators from the completed sequencing reaction usually through spin column filtration; (6) dilution of purified sequencing reaction products in formamide and separation through electrophoresis in a capillary or gel system (see Chapters 12 and 14); and (7) sequence analysis of each reaction performed and interpretation of compiled sequence information as will be described below.
DNA sequencing may be reliably performed on a variety of platforms including the ABI 310, ABI 3100, and ABI 377 (Stewart et al. 2003). These various instrument platforms will be discussed in Chapter 14. The primary difference between STR analysis and mtDNA sequencing on these multi-color fluorescence detection instruments is that a separation medium capable of single base resolution is necessary for DNA sequence analysis while it is not always needed for STR typing. Thus, the separation medium POP™-6 is commonly used for DNA sequencing while POP™-4, a less viscous and lower resolution polymer, is used for STR typing.
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