Chapter 9: Digital Analysis of Genomes Learning Objectives

Question 1

o Distinguish between digesting DNA with restriction enzymes and mechanical shearing of DNA

Answer 1

 Digesting: act of cutting genome at specific sites with restriction enzymes
 Mechanical shearing: force breaks at random locations

Question 2

o Describe how scientists produce cellular clones of recombinant DNA molecules

Answer 2

 Plasmid vector contains: origin of replication, gene conferring resistance to antibiotic, single recognition site for certain restriction enzyme
• Restriction enzyme cuts plasmid vector at restriction enzyme recognition site, opening plasmid circle; restriction enzyme makes many fragments
• Fragments have all same sticky ends
• Enzyme-cut plasmid and DNA fragments mixed together; ligase sutures them together to form circular recombinant DNA molecules

Question 3

o Explain the roles of DNA polymerase, the template, and the primer in a Sanger sequencing reaction

Answer 3

 DNA polymerase: it extends primer by adding (to 3’ end) nucleotides that are complementary to template
 Template: strand of DNA/RNA that is used as model by DNA/RNA polymerase or reverse transcriptase for creation of new complementary strand
 Primer: many copies of short sequences of DNA that are complementary to part of DNA to be sequenced

Question 4

o Compare the information that can be obtained from genomic and cDNA libraries

Answer 4

 Genomic libraries: clones in genomic library represent every locus an equal number of times; every tissue in multicellular organism can generate same genomic library; DNA fragments in genomic library carry all DNA of genome
 cDNA libraries: clones represent only fraction of genome that’s transcribed in that tissue (different tissues in a multicellular organism generate different cDNA libraries)

Question 5

o Describe how certain restriction enzymes generate DNA fragments with sticky ends, and others generate blunt-ended fragments

Answer 5

 Restriction enzymes: proteins made by bacteria that recognizes specific, short nucleotide sequences and cut DNA at those sites
 Blunt ends: restriction enzymes cuts both base pairs
 Sticky ends: restriction enzymes cuts one strand to make overhang

Question 6

o Diagram the process by which restriction enzymes and DNA ligase are used to make recombinant DNA molecules

Answer 6

 To form recombinant DNA molecules, DNA ligase links together restriction-enzyme-cut vector and genomic DNA fragments with same sticky ends

Question 7

o Describe how scientists predict the location of genes by identifying sequences conserved in the genomes of widely divergent species

Answer 7

 Scientists made three different genomic libraries that had different-sized inserts that are sheared with three different levels of energy; with smallest fragment, they made plasmid library, another plasmid library, BAC library

Question 8

o Calculate the average sizes and numbers of DNA fragments produced by digesting human genomic DNA with a given restriction enzyme

Answer 8

 The longer the sequence recognized by a restriction enzyme, the fewer but larger will be the fragments the enzyme produces when cutting genomic DNA

Question 9

o Explain why genomic DNA libraries require more colonies than are comprised by a single genome equivalent

Answer 9

 Genomic libraries contain species’ entire genome as a collection of random DNA fragments
 Multiple genome equivalents are needed to ensure representation of all parts of genome

Question 10

o Explain why a long open reading frame would suggest the existence of a protein-coding exon

Answer 10

 Exons are short, and continuing reading frame into an intron usually leads to termination sequence that close ORF

Question 11

o Summarize the process by which gel electrophoresis separates DNA fragments

Answer 11

 Pour agarose into plate with combs
 Remove comb, remove gel from plate to tank
 Power connected to tank then negative charged DNA molecule will migrate to + end
 Remove gel from tank
 Expose gel to UV light, DNA molecules will fluoresce

Question 12

o Describe the differences between the hierarchical and shotgun strategies for genome sequencing

Answer 12

 Whole-genome Shotgun Sequence: involved sequence of inserts of millions of clones selected randomly from libraries constructed with mechanically sheared genomic DNA to ensure overlap fragments; steps:
• Genomic DNA
• Male large shotgun libraries of entire genome
• Shotgun clones
• Shotgun sequence
• Assembly
 Hierarchical Strategy: separated genome into large chunks by cloning fragments in BAC vectors then they figured out the smallest overlap that incudes entire genome

Question 13

o Interpret the fluorescent peaks obtained during a DNA sequencing run as a sequence of nucleotides with the proper polarity

Answer 13

 Nested DNA fragments are separated by size on gel and detector reads color of tag at 3’ end of each fragment to determine nucleotide sequence

Question 14

o Discuss the use of reverse transcriptase in the construction of a cDNA library

Answer 14

 Retroviral RNA-dependent DNA polymerase that synthesizes DNA strands complementary
 Has the ability to use information in an RNA to generate complementary DNA

Question 15

o Describe the role of dideoxyribonucleotides in generating DNA fragments for analysis

Answer 15

 In automated DNA sequencing, chain synthesis terminates when DNA polymerase incorporates a dideoxynucleotide that has fluorescent label

Question 16

o Contrast the use of plasmid vectors with that of BAC or YAC (bacterial or yeast artificial chromosome) vectors

Answer 16

 Plasmid vectors are used for small DNA inserts

 BAC or YAC vectors can carry much larger inserts