Chapter 20 - DNA Tools and Biotechnology

Question 1

dideoxyribonucleotide chain termination sequencing

sanger method, 3 steps

Answer 1

1. DNA is denatured into two single strands
2. the strand is copied using chemically altered bases that stop each time a particular letter is incorporated into the strand (coloured tags; A, C, T, G)
3. this is carried for all four bases then the fragments are put together to reveal the sequence

Question 2

next generation sequencing (5 steps)

Answer 2

1. genomic DNA is fragmented and isolated with a bead in an aqueous solution
2. fragment is copied many times by PCR, 5’ ends are captured by the bead
3. each bead is placed in a well
4. a solution of 1 out of 4 nucleotides is added then washed off for all 4 nucleotides
5. if the next base is complementary to the nucleotide added, it will flash

Question 3

gene cloning (4 steps)

Answer 3

1. gene of interest inserted into plasmid (cloning vector) of bacterium to make recombinant DNA plasmid
2. plasmid is put into bacterial cell
3. host cell grown in culture to form clones of gene of interest cells
4. copies of gene/protein harvested for research and application

Question 4

application of gene cloning

Answer 4

• pest resistance inserted into plants
• growth hormone treats stunted growth
• alter bacteria for cleaning toxic waste
• protein dissolves blood clots in heart attack therapy

Question 5

making recombinant DNA (restriction enzyme and DNA ligase) (3 steps)

Answer 5

1. restriction enzyme cuts sugar-phosphate backbones at arrows
2. DNA fragment from other source is added (base pairing of sticky ends produces more combinations), cut by same restriction enzymes
3. DNA ligase seals the strands, recombinant plasmid is formed

Question 6

gel electrophoresis

Answer 6

1. DNA molecules placed in seperate wells, current is turned on, molecules move toward positive electrode
2. shorter molecules move faster/go further than longer molecules

Question 7

polymerase chain reaction (PCR cycle 1)

Answer 7

genomic DNA with target sequence

1. denaturation (95*C) - heat briefly to separate strands
2. annealing (55*) - cool to allow primers to form hydrogen bonds with ends of target
3. extension (70*) - DNA polymerase adds nucleotides to the 3’ end of each primer

Question 8

PCR cycle 2

Answer 8

yields 4 molecules

Question 9

PCR cycle 3

Answer 9

2 of the 8 molecules match the target sequence and are the right length

Question 10

PCR in gene cloning

Answer 10

1. PCR primers, cut with same restriction enzyme used on cloning vector
2. mix and ligate
3. recombinant DNA plasmid added to bacterial host cells, then treated with antibiotic (only cells that take up plasmid survive)

Question 11

global gene expression

Answer 11

each dot is a well containing identical copies of DNA fragments carrying a specific gene

• red well genes in one tissue bind to red cDNAs (as well as green)
• yellow in both tissues bind to both cDNAs
• black bind to neither tissue

Question 12

cDNA library

Answer 12

isolate coding sequence and follow gene expression (using reverse transcriptase)

• serves as template for PCR amplification
• run on a gel, band intensity reflects abundance of mRNA in samples