Because learning changes everything.

Chapter 10

Digital Analysis of DNA

PART III - Analysis of Genetic
Information

© McGraw Hill ©CBS Photo Archive/Getty Images 1

 CHAPTER OUTLINE

10.1 Fragmenting DNA

10.2 Cloning DNA Fragments
10.3 Sequencing DNA
10.4 Sequencing Genomes

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 Sequencing of genomes

Human Genome Project—an accurate sequence of the

human genome was initiated in 1990 and completed
(97% coverage) in 2003
By 2019, the genomes of ~8000 (eukaryotic) and ~200,000
(prokaryotic) species have been sequenced
General ideas behind genome sequencing are simple:
• Fragmenting the genome

• Cloning DNA fragments

• Sequencing DNA fragments

• Reconstructing the genome sequence from fragments

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 10.1 Fragmenting DNA

Learning Objectives:
• Distinguish between digesting DNA with restriction enzymes and
mechanical shearing of DNA

• Describe how certain restriction enzymes generate DNA fragments

with sticky ends, while others generate blunt-ended fragments

• Calculate the average sizes and numbers of DNA fragments produced

by digesting human genomic DNA with a given restriction enzyme

• Summarize the process by which gel electrophoresis separates DNA

fragments

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 Restriction enzymes fragment the genome at specific
sites

Each restriction enzyme recognizes a specific sequence of

bases anywhere within the genome
• Cuts sugar-phosphate backbones of both strands
• Restriction fragments are generated by digestion of DNA with
restriction enzymes
• Hundreds of restriction enzymes now available
Recognition sites for restriction enzymes are usually 4 – 8 bp
of double-strand DNA (see Table 10.1)
• Often palindromic – base sequences of each strand are identical when
read 5'-to-3'
• Each enzyme cuts at same place relative to its specific recognition
sequence

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 Ten commonly used restriction enzymes

TABLE 10.1 Ten Commonly Used Restriction Enzymes

Enzyme Sequence of Recognition Site Microbial Origin

TaqI Thermus aquaticus YTI

RsaI Rhodopseudomonas sphaeroides

Sau3AI Staphylococcus aureus 3A

EcoRl Escherichia coli

BamHI Bacillus amyloliquefaciens H

HindIII Haemophilus influenzae

KpnI Klebsiella pneumoniae OK8

ClaI Caryophanon latum

BssHII Bacillus stearothermophilus

NotI Nocardia otitidiscaviarum

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 Restriction enzymes produce restriction fragments with
either blunt or sticky ends

Blunt ends – cuts are

straight through both DNA
strands at the line of
symmetry
Sticky ends – cuts are
displaced equally on either
side of line of symmetry
• Ends have either 5'
overhangs or 3' overhangs

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 Different restriction enzymes produce fragments of
different length

General formula for fragment length is 4n, where n is the

number of bases in the recognition site
• 4-base recognition site occurs every 44 bp

• Average restriction fragment size is 256 bp

• 3 billion bp genome/256 = 12 million fragments

• 6-base recognition site occurs every 46 bp

• Average restriction fragment size is 4100 bp (4.1 kb)

• 3 billion bp genome/4100 = 700,000 fragments

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 Sites for three restriction enzymes in a 200 kb region of
human chromosome 11

Names and location of genes in this region are shown below

the restriction sites

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 Mechanical forces can be used to fragment DNA at
random locations

Some experiments require random cutting of DNA, not

guided cuts as with restriction enzymes
Mechanical forces can break phosphodiester bonds
• Passing DNA through a thin needle at high pressure
• Sonication (ultrasound energy)

Ends can be blunt, or may have protruding single-stranded

regions

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 Gel electrophoresis distinguishes DNA fragments
according to size 1

DNA analysis via electrophoresis, movement of charged

molecules (for example DNA fragments) in an electric field

1. Pour heated molten agarose into an acrylic plate to which

a comb has been attached, allow to cool and harden

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 Gel electrophoresis distinguishes DNA fragments
according to size 2

2. Remove comb, place gel in buffered aqueous solution, load

DNA samples into wells in gel
3. Apply electric current
• DNA has negative charge, so moves toward positive charge

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 Gel electrophoresis distinguishes DNA fragments
according to size 3

4. Remove gel from tank after electrophoresis

5. Visualize DNA fragments by

staining gel with fluorescent
dye, photograph gel under
UV light
• With linear DNA fragments,
migration distance through gel
depends on size
• Determine size of unknown
fragments by comparison of
migration to DNA markers of
known size
• Smear vs discrete bands
Supercoiled, nicked, linear, degraded forms of plasmid
© McGraw Hill ©Lee Silver, Princeton University 13
 10.2 Cloning DNA Fragments

Learning Objectives:
• Diagram the construction of recombinant DNA molecules using
restriction enzymes and DNA ligase

• Describe how scientists produce cellular clones of recombinant DNA

molecules
• Contrast the use of plasmid vectors with that of BAC or YAC (bacterial
or yeast artificial chromosome) vectors
• Explain why genomic DNA libraries require more colonies than are
contained by a single genome equivalent

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 Cloning fragments of DNA

Genomes of animals, plants, and microorganisms are too

large to analyze all at once
Molecular cloning is a means to purify a specific DNA
fragment away from all other fragments and make many
identical copies of the fragment
Two basic steps:
• Insert DNA fragments into cloning vectors to specialized chromosome-
like carriers that ensure transport, replication, and purification of DNA
inserts.
• Transport recombinant DNA into living cells to be copied
• Group of replicated DNA molecules = DNA clone

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 Creating recombinant DNA molecules with plasmid
vectors 1

Plasmid cloning vectors

have three main features:
• Origin of replication
• A selectable marker gene
(for example antibiotic
resistance)
• A synthetic polylinker, DNA
sequence containing multiple
restriction enzyme sites

Note: Bacterial artificial chromosomes (BAC) and yeast artificial

chromosomes (YAC) are alternate cloning vectors that can carry large
inserts (Plasmid ≈20 kb, BAC ≈300 kb, YAC ≈ 2000 kb)

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 Creating recombinant DNA molecules with plasmid
vectors 2

Digestion of the vector and human genomic DNA with a

restriction enzyme results in complementary sticky ends.

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 Creating recombinant DNA molecules with plasmid
vectors 3

DNA Ligase is used to seal the phosphodiester backbones

between vector and inserted fragment

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 Each genomic DNA fragment can form a different
recombinant molecule

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 Molecular cloning step 2: Host cells take up and amplify
recombinant DNA

Transformation – the process by which a cell or organism

takes up foreign DNA
• In E. coli, only 0.1% of cells
will be transformed with
plasmid
• Only cells with plasmid will
grow on media with
ampicillin
• Each cell with plasmid will
produce a colony on agar
plate, the millions of identical
plasmids in colony are a
DNA clone

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 Libraries are collections of cloned fragments

Genomic library – long-lived collection of cellular clones that

contains copies of every sequence in the whole genome inserted
into a suitable vector

Lisa Burgess/McGraw-Hill Education

Each colony contains a different recombinant plasmid, each with

a part of the human genome

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 Genomic libraries

A perfect genomic library has one copy of every sequence in

the entire genome

length of genome
 of clones in perfect library =
average size of inserts

• Genomic equivalent – number of clones in a perfect library

Impossible to obtain a perfect library
• Usually libraries are made that have four to five genomic equivalents
• Gives an average of four or five clones for each locus (95% probability
that each locus is present at least once)

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 10.3 Sequencing DNA

Learning Objectives:
• Explain the roles of DNA polymerase, the template, and the primer in a
Sanger sequencing reaction

• Describe the role of dideoxyribonucleotides in generating DNA

fragments for analysis
• Interpret the fluorescent peaks obtained during a DNA sequencing run
as a sequence of nucleotides with the proper polarity

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 Sanger sequencing uses DNA polymerase to
make new DNA

DNA polymerase requires:

• Template— single strand of DNA to copy

• Deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP), basic

building blocks for new DNA
• Primer—short single stranded DNA molecule that is complementary to
part of the template, includes free 3’ end to which DNA polymerase
can attach new nucleotides

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 A recombinant plasmid is a good template for Sanger
sequencing

• Cloned recombinant DNA is

denatured (heat breaks H
bonds between strands)
• Strands are mixed with an
oligonucleotide primer
(made in DNA synthesizer)
~20 bp complementary to
template strand
• As temperature is lowered,
primers and template strand
anneal (hybridize)

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 Sanger sequencing generates a series of single-
stranded DNA fragments

Hybridized template and primers are

mixed with DNA polymerase, dNTPs,
and small amounts of
dideoxyribonucleotide triphosphates,
ddNTPs (each with unique
fluorescent tag)

• ddNTPs lack a 3’ –OH, halting

polymerization

Result is a set of nested fragments,

with a different 3’ end

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 Incorporation of a dideoxynucleotide (ddNTP) terminates
DNA synthesis

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 Nested fragments are separated by size using
electrophoresis

A special gel separates DNA fragments that differ in size by

only one nucleotide
• Smaller DNA fragments migrate quickly, appear at the bottom of the
gel

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 Each ddNTP is labeled with a different fluorescent dye
for detection of the sequence

Each lane displays the

sequence obtained from a
separate DNA sample and
primer
Each fragment terminates
with a specific ddNTP labeled
with a unique fluorescent dye
• DNA fragments are
electrophoresed based on size,
color of terminal ddNTP is
recorded to identify nucleotide
code

© McGraw Hill (e): ©Jean Claude Revy/Phototake; 29

 Automated DNA sequencing

Fragments flow past a laser beam and the color of the

terminal base is digitally recorded

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 DNA sequence trace and inferred DNA sequence from
automated Sanger sequencing

• Computer reads of sequence complementary to the template

strand
• Sequence is read from left to right (5'-to-3' synthesis from primer)

© McGraw Hill (f): Courtesy of Joshua J. Filter, Cornell University, Ithaca, New York 31
 10.4 Sequencing Genomes

Learning Objectives:
• Explain why overlap between individual DNA sequences is required to
reconstruct the sequence of a genome

• Describe how paired-end sequencing allows genome projects to

overcome the problems presented by the presence of repetitive DNA

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 Genome comparisons

TABLE 10.2 Genome Comparisons

Organism Organism
Number of Number of Genome
Type Species Chromosomesa Genesb Size (Mb)
Bacterium Escherichia coli 1 ∼4400 4.6d
Yeast Saccharomyces cerevisiae 16 ∼6000 12.5
Worm Caenorhabditis elegans 6 ∼22,000 100.3
Fly Drosophila melanogaster 4 ∼17,000 122.7
Mustard weed Arabidopsis thaliana 5 ∼28,000 135
Mouse Mus musculus 20 ∼28,000 2,700
Human Homo sapiens 23 ∼28,000 3,300
Lungfish Protopterus aethiopicus 14 ?? 133,000
Canopy plant Paris japonica 5c ?? 152,400
aHaploid chromosome complement except where indicated.
bIncludes non-protein-coding genes.
cThis species is an octoploid; 5 is the basic chromosome number (see Chapter 13).
dE. coli genomes vary in size; 4.6 Mb is a representative length (see Chapter 14.)

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 The Human Genome Project began using the
hierarchical strategy

1. Construct BAC genomic library

2. Identify sets of overlapping BAC clones
3. Shear DNA from each BAC separately to make smaller
clones
4. Sequence DNA
5. Assemble sequences based on overlap
Strategy was successful, but labor-intensive, inefficient and
expensive

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 Celera developed the whole-genome shotgun strategy
for genome sequencing

1. Create genomic library of

overlapping fragments in
plasmid vectors

2. Sequence DNA inserts of

randomly chosen library
plasmids (”shotgun”)

3. Assemble sequences based

on overlap of sequences into
contigs – continuous base
pair sequences

The whole-genome shotgun approach can be highly automated

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 Repeats prevent correct assembly of single shotgun
sequence reads

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 Each shotgun clone was partially sequenced from
both ends

Paired-end sequencing,
sequencing a BAC clone
library rather than smaller
inserts of plasma clones
Each BAC clone insert
gave three pieces of
information:
• Two ~1000 bp sequence read
• Knowledge that the two
sequences were ~200-300 kb
apart

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 Paired-end sequences direct correct assembly of unique
sequences flanking repeats

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