Chapter 18_Recombinant DNA Technology

Question 1

DNA Sequencing

Answer 1

This method enables researchers to determine the base sequence of DNA found in genes and other chromosomal regions.

Question 2

Dideoxy Sequencing

Answer 2

Developed by Frederick Sanger and colleagues. It has become the more popular method of DNA sequencing.

Question 3

DNA polymerase connects adjacent deoxyribonucleotides by…

Answer 3

…catalyzing a covalent bond between the 5’ phosphate one one nucleotide and the 3’ -OH group on the previous nucleotide.

Question 4

Describe Dideoxy Sequencing

Answer 4

• Synthesize deoxyribonucleotides that are missing the -OH group at the 3’ position. These synthetic nucleotides are called dideoxyribonucleotides (ddNTPs).
  
  • If a ddNTP is added to a growing DNA strand, the strand can no longer grow because the ddNTP is added to a growing DNA strand, the strand can no longer grow because the ddNTP is missing the 3’ -OH group. The incorporation of a ddNTP into a growing strand is called a CHAIN TERMINATION.

Question 5

Compare DNA strand labeling before and today.

Answer 5

• Used to label newly made DNA with radioisotopes
• Today we use automated DNA sequencing in which each type of ddNTP is labeled with a different colored fluorescent molecule.

Question 6

What is the common way to fluorescently label ddNTPs?

Answer 6

• ddA is green
• ddT is red
• ddG is yellow
• ddC is blue

Question 7

Do you need a small or large amount of segmented DNA to sequence? If you do need more, how is it obtained?

Answer 7

Prior to automated DNA sequencing, the segment of DNA to be sequenced must usually be obtained in large amounts. This is accomplished by gene cloning.

Question 8

What are the steps of DNA sequencing?

Answer 8

• A sample containing many copies of the single stranded DNA is mixed with many primers that will bind to the primer annealing site.
• All four types of deoxyribonucleotides and DNA polymerase are then added to the annealed DNA fragments. The tube also has a low concentration of each ddNTP.
• Tube is incubated to allow DNA polymerase to make strands complementary to the target DNA sequence.
• DNA synthesis continues until a ddNTP is incorporated into a growing strand.
• DNA strands are separated according to lengths by running them on a slab gel or more commonly by running them through a gel filled capillary tube. Shorter strands move to bottom of gel.

Question 9

Sequencing Ladder

Answer 9

Reading the base sequence from bottom to top of the of the gel.

Question 10

Site-Directed Mutagenesis

Answer 10

Allows a researcher to produce a mutation at a specific site within a cloned DNA segment.

Question 11

Explain how site directed mutagenesis works

Answer 11

• An oligonucleotide primer is allowed to hybridize or anneal to the template DNA. The primer is synthesized chemically.
  
  • 1. Most of the sequence of the primer is complementary to the site in the DNA where the mutation is to be made.
  • 2. The primer contains a region of mismatch where the primer and template DNA are not complementary.
• After the primer and template have annealed, the complementary strand is synthesized by adding deoxyribonucleoside triphosphates (dNTPs), DNA polymerase, and DNA ligase. This yields a double stranded molecule that contains a mismatch only at the desired location. This can then be introduced into a bacterial cell.
• The DNA mismatch is likely to be repaired. Depending on which base is replaced, this may produce the mutant or original sequence.

Question 12

Site-Directed Mutagenesis

Answer 12

Allows a researcher to produce a mutation at a specific site within a cloned DNA segment.

Question 13

Explain how site directed mutagenesis works

Answer 13

• An oligonucleotide primer is allowed to hybridize or anneal to the template DNA. The primer is synthesized chemically.
  
  • 1. Most of the sequence of the primer is complementary to the site in the DNA where the mutation is to be made.
  • 2. The primer contains a region of mismatch where the primer and template DNA are not complementary.
• After the primer and template have annealed, the complementary strand is synthesized by adding deoxyribonucleoside triphosphates (dNTPs), DNA polymerase, and DNA ligase. This yields a double stranded molecule that contains a mismatch only at the desired location. This can then be introduced into a bacterial cell.
• The DNA mismatch is likely to be repaired. Depending on which base is replaced, this may produce the mutant or original sequence.

Question 14

Polymerase Chain Reaction

Answer 14

(PCR) A way to copy DNA without the aid of vectors and host cells. It is used to make large amounts of DNA in a defined region that is flanked by two primers.

Question 15

Primer

Answer 15

Oligonucleotides (short segments of DNA about 15 to 20 nucleotides in length).

Question 16

What is the end result of PCR?

Answer 16

The region that is flanked by the primers, which contains the gene of interest, is amplified. (Amplification means many copies of the region have been made). Basically, the region between the two primers has been cloned.

Question 17

What are the reageants needed for PCR?

Answer 17

Primers, template DNA (sample of DNA that contains a gene of interest), deoxyribonucleoside triphosphates (dNTPs), and a thermostable form of DNA polymerase such as Taq polymerase (isolated form the bacterium Thermus aquaticus).

Question 18

Why is a thermostable form of DNA polymerase necessary in PCR?

Answer 18

Because PCR involves heating steps that inactivate most other natural forms of DNA polymerase.

Question 19

What are the three steps of PCR?

Answer 19

• 1. Denaturation: To make copies of DNA, template DNA is denatured by heat treatment, causing strands to separate.
• 2. Primer Annealing: As temp is lowered, oligonucleotide primers bind to the DNA in a process called annealing.
• 3. Primer Extension: Temp is raised slightly, and Taq polymerase catalyzes the synthesis of complementary DNA strands in the 5’ to 3’ direction, starting at the primers. This doubles the amount of template DNA.

Question 20

Specific, semispecific, and nonspecific PCR

Answer 20

• Specific: Can amplify a particular region of DNA from a very complex mixture of template DNA.
• Semispecific: The primers recognize a known repetitive DNA sequence found at several sites within the genome.
• Nonspecific: Uses a mixture of short PCR primers with many different random sequence.s These primers anneal randomly throughout the genome and amplify most of the chrmosomal DNA.

Question 21

Reverse Transcriptase PCR

Answer 21

RNA is isolated from a sample and mixed with deoxynucleotides, reverse transcriptase, and a primer that binds near the 3’ end of the RNA of interest. This generates a single stranded cDNA, which then can be used as template DNA in a conventional PCR reaction. The end result is that the RNA has been amplified to produce many copies of DNA.