Chapter 9.3: Sequencing DNA

Question 1

What are the 3 things DNA polymerase requires?

Answer 1

 A template; a single strand of DNA to copy  Deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) that are the basic building blocks for newly synthesized DNA  A primer; a short single-stranded DNA molecule (an oligonucleotide) that is complementary to part of the template and that provides the 3’ end to which DNA polymerase can attach new nucleotides

Question 2

What is a read (Sanger sequencing)?

Answer 2

o The computers in DNA sequencing machines also have base-calling software that interpret the peaks as specific bases and that generate a digital file, called a read, of the sequence of As, Cs, Gs, and Ts comprising the newly synthesized DNA

Question 3

Describe step 1: hybridization of template and primer in automated sanger sequencing

Answer 3

 A cloned recombinant DNA is denatured (melted into single strands), using heat to break apart the hydrogen bonds joining the two strands. One of these strands serves as the template. The recombinant DNA is mixed with an oligonucleotide primer (previously made in a DNA synthesizer) whose sequence is complementary to about 20 bases in the vector portion of the template strand. As the temperature is lowered, the template and primer anneal(hybridize) together.

Question 4

Describe step 2: generating a nested set of polymerization products of automated sanger sequencing

Answer 4

 The hybrid template-primer is now mixed with DNA polymerase, large amounts of the four deoxynucleotide triphosphates (dNTPs), and smaller amounts of the four dideoxynucleotide triphosphates (ddNTPs). Each ddNTP has a fluorescent tag of a different color. DNA polymerase synthesizes a new strand of DNA complementary to the template by adding nucleotides sequentially onto the 3’ end of the primer. Synthesis terminates when a dideoxynucleoside is added to the chain. The reaction generates a nested set of products each with the same 5’ end but a different 3’ end. The terminating dideoxynucleotide at the 3’ end color codes each product. After melting apart the newly synthesized DNA from the template, these products are electrophoresed on a special gel that can separate DNAs that differ in size by a single nucleotide. As each fragment moves past a laser beam, the color of the terminal base is detected and recorded.

Question 5

Define hybridization (sanger sequencing)

Answer 5

o Sanger sequencing allows the template and primer to interact through the process for hybridization: that natural tendency of complementary single-stranded molecules of DNA or RNA to base pair and form double helixes