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TRANSCRIPT
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CHAPTER 8
DNA: The Chemical Nature of the GeneKey Characteristics of Genetic Material
• Genetic material must contain complex information
• Genetic material must replicate faithfully
• But also must have the capacity to vary
• Genetic material must encode the phenotype.
All Genetic Information Is Encoded in
the Structure of DNA or RNA
• Early studies of DNA
• Miescher: Nuclein
• Kossel: DNA contains four nitrogenous bases
• Chargaff’s rules
• DNA as the source of genetic information
– Identification of the transforming principle
• Griffith experiment
• The experiment by Avery, Macleod, and McCarty
• The Hershey–Chase experiment
Chargaff’s
rules
Griffith
Transforming principle
Avery, McCleod, McCarty
• Treat with enzymes
• DNase
• RNase
• Protease
DNA is transforming principle
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Hershey, Chase
• DNA and Protein w/ radioactive
isotopes
• Phosphorus on DNA
• Sulfur on protein
Hershey, Chase
• DNA and Protein w/ radioactive
isotopes
• Phosphorus on DNA
• Sulfur on protein
• Bacteria w protein coat
removed were radioactive
• So were some of progeny
• DNA is the genetic material
Genetic Information Is Encoded in the
Structure of DNA or RNA
• DNA as the source of genetic information
– Watson and Crick’s discovery of the three-
dimensional structure of DNA
– X-ray diffraction image of DNA
DNA Consists of Two Complementary and
Antiparallel Nucleotide Strands that Form
a Double Helix
• The primary structure of DNA
– Deoxyribonucleotides
• Nucleotides
– Three parts: sugar, a phosphate, and a base
» Purine or pyrimidine base:
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Concept Check
How do the sugars of RNA and DNA differ?
a. RNA has a six-carbon sugar; DNA has a five-
carbon sugar
b. The sugar of RNA has a hydroxyl group that is not
found in the sugar of DNA
c. RNA contains uracil; DNA contain thymine
d. DNA’s sugar has a phosphorus atom; RNA’s sugar
does not
DNA Consists of Two Complementary and
Antiparallel Nucleotide Strands that Form
a Double Helix
• Secondary structures of DNA
– The double helix
– Backbone formed through phosphodiester
bonds
– Hydrogen bond and base pairing
– Antiparallel complementary DNA strands
DNA and RNA
DNA
• Sugar/phosphate backbone
• Double stranded
• One Fewer O on sugar
• Strands run antiparallel
• 5’ (5’-carbon to free phosphate)
• 3’ (3’-carbon to free OH)
• Complimentary strands
• Bound by Hydrogen bonds
• T-A (2 bonds)
• C-G (3 bonds)
• Helical
RNA
• Sugar/phosphate backbone
• Single stranded
• Often folds over on self
• OH on sugar
Concept Check
The antiparallel nature of DNA refers to
a. its charged phosphate groups.
b. the pairing of bases on one strand with bases on
the other strand.
c. the formation of hydrogen bonds between bases
from opposite strands.
d. the opposite direction of the two strands of
nucleotides.
• Secondary structures of DNA
– Three-dimensional structure identified by
Watson and Crick refers to B-DNA
– Different secondary structures
DNA Consists of Two Complementary and
Antiparallel Nucleotide Strands that Form
a Double Helix
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Large Amounts of DNA Are Packed
into a Cell
• DNA must be tightly packed to fit in small
spaces
• Supercoiling
– Positive supercoiling,
– Negative supercoiling
– Topoisomerase: The enzyme responsible for
adding and removing turns in the coil
The Bacterial Chromosome
• Most bacterial genomes have a single
circular DNA molecule
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Chromatin – Eukaryotic organisms
Chromatin - DNA and Proteins
• Euchromatin
• active
• Heterochromatin
• Inactive (always wrapped)
• Histones – most common protein in chromatin
• DNA wrapped around histones
• Positively charged - attracts Negative DNA
• Nucleosome – beads on a string
• DNA wrapped around 8 histones (2 of each 2A,2B, 3 and 4)
• H1 – acts as spacer (maybe does more)
• Fibers – formed by packing nucleosomes together
DNA Packed into a Cell
Beads attached to about 200 bp
• Chromatin structure
– Changes in chromatin structure
• Epigenetic changes: methylation; capable of being
reversed and often due to environmental factors
DNA Packed into a Cell
Epigenetic
changes: DNA
methylation
causes different
coat colors in
mice
Eukaryotic Chromosomes Possess
Centromeres and Telomeres
• Centromeres
• Constricted portion
• Binding site of
kinetochore (site of
spindle attachment)
• Tandem repeats
• Telomeres
• End of chromosome
• Repeated units
• CCC and GGGs
• Looped over
• Protection
Eukaryotic DNA Contains Several
Classes of Sequence Variation
• Organisms differ in amount of DNA per
cell (C value)
Types of DNA sequences in eukaryotes
– Unique sequence DNA
• Genes that encode proteins
• Gene family: Similar but not identical copies of unique DNA
sequences that arose through duplication of an existing gene
– Repetitive DNA
• Moderately repetitive DNA: 150 ~ 300 bp long
– Tandem repeat sequences
– Interspersed repeat sequences
– Highly Repetitive DNA
• Highly repetitive DNA: less than 10 bp long
– Microsatellite DNA
Eukaryotic DNA Contains Several
Classes of Sequence Variation
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Concept Check
Most of the genes that encode proteins are
found in
a. unique-sequence DNA.
b. moderately repetitive DNA.
c. highly repetitive DNA.
d. All of the above.
GENETICS ESSENTIALS
Concepts and ConnectionsTHIRD EDITION
Benjamin A. Pierce
CHAPTER 9
DNA Replication and Recombination
© 2014 W. H. Freeman and Company
• Replication has to be accurate
– One error per million bp leads to 6400
mistakes every time a cell divides, which
would be catastrophic
• Replication also takes place at high speed
– E. coli replicates its DNA at a rate of 1000
nucleotides per second
Genetic Information Must Be Accurately
Copied Every Time a Cell Divides
DNA Replication Takes Place in a
Semiconservative Manner
• Proposed DNA Replication Models
– Conservative replication model
– Dispersive replication model
– Semiconservative replication
• Meselson and Stahl’s Experiment
– Two isotopes of nitrogen
• 14N common form; 15N rare heavy form
• E.coli were grown in a 15N media first, then
transferred to 14N media
• Cultured E.coli were subjected to equilibrium
density gradient centrifugation
DNA Replication Takes Place in a
Semiconservative Manner
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• Modes of Replication
– Replicons: Units of replication
• Replication origin
– Theta replication: circular DNA, E. coli; single
origin of replication forming a replication fork,
and it is usually a bidirectional replication
DNA Replication Takes Place in a
Semiconservative Manner
Linear eukaryotic replication
– Eukaryotic cells
– Multiple origins - replicons
– A typical replicon: ~ 200,000-300,000 bp in
length.
• Requirements of replication• A template strand
• Raw material: nucleotides
• Enzymes and other proteins
DNA Replication Takes Place in a
Semiconservative Manner
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Linear eukaryotic replication
• Direction of Replication
– DNA polymerase add nucleotides only to the
3 end of growing strand
– The replication can only go from 5 3
– Continuous and discontinuous replication
DNA Replication Cont.
Linear eukaryotic replication
• Direction of replication
• Leading strand: undergoes continuous replication
• Lagging strand: undergoes discontinuous
replication
• Okazaki fragment: the discontinuously synthesized
short DNA fragments forming the lagging strand
DNA Replication Cont.
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Concept Check 2
Discontinuous replication is a result of
which property of DNA?
a. Complementary bases
b. Antiparallel nucleotide strands
c. A charged phosphate group
d. Five-carbon sugar
Bacterial Replication Requires a Large
Number of Enzymes and Proteins
• Bacterial DNA Replication
– Initiation
• oriC (single origin replicon)
• an initiation protein (DnaA in E.coli)
– Unwinding
• DNA helicase
• Single-strand-binding proteins (SSBs)
• DNA gyrase (topoisomerase)
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Replication Cont.
Order used in the course of replication:
Initiator proteins – bind to origin of replication
Helicase - unwinds
Single-strand-binding protein - detach
DNA gyrase – releases stress on molecule
Then elongation
• Elongation
– Primers: an existing group of RNA nucleotides
with a 3-OH group to which a new nucleotide
can be added. It is usually 10 to 12
nucleotides long.
– Primase: RNA polymerase
Bacterial Replication Requires a Large
Number of Enzymes and Proteins
• Elongation: carried out by DNA polymerase III
• Removing RNA primer: DNA polymerase I
‒ Connecting nicks after RNA primers are removed:
DNA ligase
• Termination: when two replication forks meet
or by a specific termination sequence
Replication Cont.
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Bacterial
polymerasesDNA ligase links
Okazaki fragments
Video: 0903
Looping:
• Leading strand and lagging
strand with DNA polymerase
complex
The fidelity of DNA replication
• Errors every 100,000 nucleotides
• Proofreading: DNA polymerase I : 3 5
exonuclease activity removes the incorrectly paired
nucleotide, reduces errors to 1 in 10 million
• Mismatch repair: corrects errors after replication is
complete (more later): Errors reduced even more
Replication Cont.
• Eukaryotic DNA Replication
– Origin-recognition complex (ORC) binds to
origins and initiates DNA replication
• Eukaryotic DNA polymerases
• Replication at the ends of chromosomes‒ Telomeres and telomerase
Eukaryotic DNA Replication Is Similar to
Bacterial Replication but Differs in
Several Aspects
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Many DNA polymerases in eukaryotic cells
Important details
• Multiple origins
• Origin complex
• ORC
• RNA Primers
• DNA nucleotides
• Gap – no opposite
strand
Gap filled, or partially
filled with the help of
telomerase
Gap filled, or partially
filled with the help of
telomerase
Recombination Takes Place Through the
Breakage, Alignment, and Repair of DNA
Strands
• Homologous recombination: exchange is
between homologous DNA molecules
during crossover
• Holliday junction and single-strand break
• The double-strand break model of
recombination
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Death cap mushroom: kills by inhibiting RNA polymerase II
CHAPTER 10
Transcription
GENETICS ESSENTIALS
Concepts and Connections
THIRD EDITION
RNA, a Single Strand of Ribonucleotides,
Participates in a Variety of Cellular Functions
• RNA: evidence suggests RNA was original
genetic material
• The structure of RNA
– Primary structure
– Secondary structure
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Transcription Is the Synthesis of an RNA
Molecule from a DNA Template
The template
– The transcribed strand: template strand
– The transcription unit
• A promoter
• RNA-coding sequence
• Terminator
Sequence forms from one side – template strand – but different
genes may have opposite template strands. Terminology:
sometimes sense strand(+) anti-sense strand (-).
Transcriptional Unit – RNA and other regions
• Promoter – binding site starting transcription
• Coding region - RNA sequence from here
• Terminator – Signal that ends transcription
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The substrate for transcriptionRibonucleoside triphosphates—rNTPs added to the 3 end of the RNA molecule
Transcription Is the Synthesis of RNA
Molecule from a DNA Template
To Know:
Initiation
– The substrate for transcription:
Ribonucleoside triphosphates—rNTPs added
to the 3 end of the RNA molecule
– The transcription apparatus: Eukaryotic RNA
polymerases
Transcription Consists of Initiation, Elongation,
and Termination
Transcription Consists of Initiation, Elongation,
and Termination
Initiation
• Bacterial promoters
Consensus sequences: sequences that possess
considerable similarity.
• –10 consensus: 10 bp upstream of the start site
• Pribnow box:
– 5 TATAAT 3
– 3 ATATTA 5
• –35 consensus sequence: TTGACA
Consensus sequence
similar enough so
binding molecule can
bind.
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• Initiation
– Initial RNA synthesis: no primer is required
– The location of the consensus sequence
determines the position of the start site
• Elongation
– RNA elongation is carried out by the action of
RNA polymerase
Transcription Consists of Initiation,
Elongation, and Termination
Holoenzyme is just a term for molecule formed by enzyme and co-enzyme
Concept Check
What is the function of the sigma factor?
The sigma factor controls the binding of RNA
polymerase to the promoter by forming the
holoenzyme.
• Elongation: the molecular structure of
eukaryotic RNA polymerase II and how it
functions during elongation has been revealed
through the work of Roger Kornberg and his
colleagues
• Termination:
– RNA polymerase I
– RNA polymerase II
– RNA polymerase III
Transcription Consists of Initiation, Elongation,
and Termination
Termination
– Rho-dependant termination: uses rho factor
– Rho-independent termination: hairpin
structure formed by inverted repeats, followed
by a string of uracils
Transcription Consists of Initiation,
Elongation, and Termination
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RNA termination
• Nucleotides are added to the RNA molecule until it transcribes
a terminator
• Activity mostly known from bacteria
• Rho-dependent
• Inverted sequence forms hairpin
• Poly Us at end cause pause
• Rho protein unwinds DNA/RNA
• Polycistronic (uncommon in eukaryotes)
• Single terminator for several genes
• Will not test on the details of termination
Many Genes Have Complex Structures
• Gene Organization• The concept of colinearity and noncolinearity
• Number of nucleotides in a gene should be
proportional to the number of amino acids in the
encoded protein
• DNA much longer than mRNA; demonstrated through
hybridization
1958 idea - Crick
• DNA sequence
corresponds to protein
sequence
• Colinear
• But DNA is much longer
than the RNA is produces
Collinearity is not the case
Loops when DNA is re-annealed with
complementary RNA indicates nucleotides
not paired
Non-coding region of DNA = loops
Exons = coding
Introns = non-coding
This concept is important
Many Genes Have Complex Structures
•The concept of the gene revisited
•The gene includes:
• DNA sequences that code for all exons and
introns
• Those sequences at the beginning and end of
the RNA that are not translated into a protein,
including the entire transcription unit– The promoter
– The RNA coding sequence
– The terminator
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Many RNA Molecules Are Modified After
Transcription in Eukaryotes
• A mature mRNA contains 5′ untranslated region (5′ UTR, or leader sequence)
◦ Shine–Dalgarno sequence
• Protein-coding region
• 3′ untranslated region
Many RNA Molecules Are Modified After
Transcription in Eukaryotes
• The addition of the 5′ cap
‒ A nucleotide with 7-methylguanine; 5′-5′ bond is
attached to the 5′-end of the RNA
• The addition of the poly(A) tail
‒ ~ 50 to 250 adenine nucleotides are added to the
3′-end of the mRNA
Many RNA Molecules Are Modified After
Transcription in Eukaryotes
• RNA splicing
‒ Consensus sequences
• 5′ consensus sequence: GU A/G AGU: 5′ splice
site
• 3′ consensus sequence: CAGG
‒ The process of splicing
RNA processing
• mRNA modified before translation to amino acids
• Cleavage cuts out introns
• Adenine tail added
Ends of exons spliced together
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RNA processing
• 5’ cap – more later
• Poly A tail – slows degradation of RNA
Transfer RNA (tRNA)
• The structure and processing of transfer
RNA (tRNA)
• Common secondary structure—the
cloverleaf structure
• Anticodon
Ribosomal RNA (rRNA) and the Ribosome
• The structure of the ribosome
– Large ribosome subunit
– Small ribosome subunit
Regulation via RNAs
MicroRNAs
• Inhibit translation
Small interfering RNAs
• Degrade mRNA
GENETICS ESSENTIALS
Concepts and ConnectionsTHIRD EDITION
Benjamin A. Pierce
CHAPTER 11
From DNA to Proteins: Translation
© 2014 W. H. Freeman and Company
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The Genetic Code Determines How the Nucleotide
Sequence Specifies the Amino Acid Sequence of a
Protein
• Breaking the genetic code
• The reading frame and initiation codons
• Termination codons
• The universality of the code
• Breaking the genetic code
• Codon: a triplet RNA code
• 64 possible codons‒ 3 stop codons
‒ 61 sense codons
The Genetic Code
• Synonymous codons: codons that specify the same amino acid
• Isoaccepting tRNAs: different tRNAs that accept the same amino acid but have different anticodons
• Codons
• Sense codons: encoding amino acid
• Initiation codon: AUG
• Termination codon: UAA, UAG, UGA
• Wobble hypothesis
The Genetic Code
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Concept Check
Through wobble, a single can pair with more
than one .
a. codon; anticodon
b. group of three nucleotides in DNA; codon in
mRNA
c. tRNA; amino acid
d. anticodon; codon
•The Reading Frame and Initiation
Codons• Reading frame: Read in groups of three so the
starting point is very important.
• Nonoverlapping: A single nucleotide may not be
included in more than one codon.
• The universality of the code: near universal, with
some exceptions
11.1 The Genetic Code Determines How the
Nucleotide Sequence Specifies the Amino Acid
Sequence of a Protein
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Amino Acids Are Assembled Into a
Protein Through Translation
• The binding of amino acids to transfer RNAs
• The initiation of translation
• Elongation
• Termination
• Amino acids bind to tRNAs• The specificity between an amino acid and its tRNA is
determined by each individual aminoacyl-tRNA synthesis
• There are 20 different aminoacyl-tRNA syntheses in a cell
• The initiation of translation
• Initiation factors IF-3, initiator tRNA with N-
formylmethionine attached to form fmet-tRNA
• Energy molecule: GTP
Translation
•Amino acids bind to tRNAs•The specificity between an amino acid and its
tRNA is determined by each individual
aminoacyl-tRNA synthesis
•There are 20 different aminoacyl-tRNA
syntheses in a cell
• The Initiation of Translation
• The Shine–Dalgarno consensus sequence in
bacterial cells is recognized by the small unit
of ribosome.
• The Kozak sequence in eukaryotic cells
facilitates the identification of the start codon.
11.2 Amino Acids Are Assembled into a
Protein Through Translation
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• Elongation
• Exit site E
• Peptidyl site P
• Aminoacyl site A
• Elongation factors: Tu, Ts, and G
Translation
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• Termination
• Termination codons: UAA, UAG, and UGA
• Release factors (RFs)
Translation
Mini video
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• Polyribosomes: an mRNA with several
ribosomes attached
Additional Properties
• Proteins go through modifications after
translation
• Folding
• Most proteins must fold into a 3D shape in order to
be functional
• Molecular chaperones
• Molecules that prevent incorrect folding
Additional Properties, Proteins
GENETICS ESSENTIALS
Concepts and ConnectionsTHIRD EDITION
Benjamin A. Pierce
CHAPTER 14
Molecular Genetic Analysis and
Biotechnology
© 2014 W. H. Freeman and Company
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Techniques of Molecular Genetics Have
Revolutionized Biology
• Recombinant DNA Technology (Genetic Engineering)– Techniques for locating, isolating, altering, and
studying DNA segments
• The Molecular Genetics Revolution– Biotechnology: the use of these techniques to
develop new products
• Working at the Molecular Level
Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• First step: isolate DNA segment or gene
• Cutting and joining DNA fragments—restriction
enzymes
• Viewing DNA fragments
• Locating DNA fragments with Southern blotting
and probes
Cutting and Joining DNA
Fragments
• Restriction enzymes: recognizing and cutting
DNA at specific nucleotide sequences
• Type II restriction enzyme: most useful enzyme
• By adding methyl groups to the recognition
sequence to protect itself from being digested by
its own enzyme in bacteria
Cutting and Joining DNA
Fragments
• Cohesive ends: fragments with short, single-
stranded overhanging ends
• Blunt ends: even-length ends from both single
strands
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Viewing DNA
Fragments• Gel electrophoresis
• Autoradiography
Concept Check
DNA fragments that are 500 bp, 1000 bp, and
2000 bp in length are separated by gel
electrophoresis. Which fragment will migrate
farthest in the gel?
a. The 2000-bp fragment.
b. T he 1000-bp fragment.
c. The 500-bp fragment.
d. All fragments will migrate equal distances.
Locating DNA Fragments with
Southern Blotting and Probes
• Probe: DNA or RNA with a base sequence
complementary to a sequence in the gene of
interest
Southern blot named after Edwin Southern, similar methods named after take
off on directional aspect: Western blot, Northern Blot, Eastern blot.
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Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
• Cloning genes
• Application: the genetic engineering of plants
with pesticides
• Amplifying DNA fragments with the polymerase
chain reaction
Cloning Genes
• Gene cloning: amplifying a specific piece of
DNA via a bacteria cell
• Cloning vector: a replicating DNA molecule
attached with a foreign DNA fragment to be
introduced into a cell
Cloning Genes
• Plasmid vectors
– Plasmids: circular DNA molecules from bacteria
– Insert foreign DNA into plasmid using restriction
enzymes
– Linkers: synthetic DNA fragments containing
restriction sites
• Transformation of host cells with plasmids
• Screening cells for recombinant plasmids
– Selectable markers are used to confirm whether the
cells have been transformed
Cloning Genes
• Other gene vectors:
– Cosmids
– Bacterial artificial chromosomes (BACs)
– Yeast artificial chromosome
– Plasmid
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Concept Check 3
How is a gene inserted into a plasmid cloning
vector?
The gene and plasmid are cut with the same
restriction enzyme and mixed together. DNA ligase
is used to seal nicks in the sugar–phosphate
backbone.
Amplifying DNA Fragments with the
Polymerase Chain Reaction (PCR)
• The PCR reaction
– Taq polymerase: stable DNA polymerase at high
temperature
– Reverse-transcription PCR
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Amplifying DNA Fragments with the
Polymerase Chain Reaction (PCR)
• Limitations of PCR
– Prior knowledge of target DNA
– Contamination
– Accuracy
– Amplified fragments are less than 2 kb
Amplifying DNA Fragments with the
Polymerase Chain Reaction (PCR)
• Applications of PCR
– Real-time PCR: quantitatively determining the
amount of DNA amplified as the reaction proceeds
Molecular Techniques Can Be Used to Find
Genes of Interest
• Gene libraries
• In situ hybridization
• Positional cloning
• Application: isolating the gene for cystic fibrosis
Gene Libraries
• DNA library: a collection of clones containing all the DNA fragments from one source
– Creating a genomic DNA library
– cDNA libraries: consisting only of those DNA sequences that are transcribed into mRNA
Gene Libraries
• Screening DNA libraries
– Plating clones of the library
– Probing plated colonies or plaques
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Positional Cloning
• Isolating genes on the basis of their position on
a genetic map
• Chromosome walking
• Chromosome jumping• Larger steps “jump” around
14.3 DNA Sequences Can Be Determined
and Analyzed
• Restriction Fragment Length Polymorphisms
(RFLPs)
• DNA sequencing
• Next-generation sequencing technologies
• DNA fingerprinting
Restriction Fragment Length Polymorphisms
• Some DNA fragments have different restriction
sites due to mutation for the same restriction
enzyme
• Causes polymorphisms within a population
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DNA Sequencing
• Sanger’s dideoxy-sequencing method
– Dideoxyribonucleoside triphosphate (ddNTP)
lacks a 3′-oh group, which terminates DNA
synthesis
No 3’-OH group
Terminates synthesis
Concept Check 4
In the dideoxy-sequencing reaction, what
terminates DNA synthesis at a particular base?
a. The absence of a base on the ddNTP halts the
DNA polymerase
b. The ddNTP causes a break in the sugar–phosphate
backbone
c. DNA polymerase will not incorporate a ddNTP into
the growing DNA strand
d. The absence of a 3’-OH group
Next-Generation Sequencing Technologies
• Pyrosequencing
• Illumina sequencing
• Third-generation sequencing
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DNA Fingerprinting (DNA Profiling)
• Microsatellites: (short tandem repeats, STRs)
variable number of copies of repeat sequences
possessed by many organisms
• Detected by PCR
• Fragments represented as peaks on a graph
– Homozygotes: single tall peak
– Heterozygotes: two shorter peaks
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14.4 Molecular Techniques Are Increasingly
Used to Analyze Gene Function
• Forward and reverse genetics
• Transgenic animals
• Knockout mice
Forward and Reverse Genetics
• Forward genetics: Begins with a phenotype to
a gene that encodes the phenotype
• Reverse genetics: Begins with a gene of
unknown function, first inducing mutations
and then checking the effect of the mutation
on the phenotype
Transgenic Animals
• An organism permanently altered by the addition
of a DNA sequence to its genome
• Transgene
Knockout Mice
• A normal gene of the mouse has been fully
disabled
• Knock-in mice: a mouse carries an inserted
DNA sequence at specific locations
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Biotechnology Harnesses the Power
of Molecular Genetics
• Pharmaceutical products
• Specialized bacteria
• Agriculture products
• Genetic testing
• Gene therapy
Potential Benefits of genetically
Engineered plant?• Pest resistant
• Increased yield
• Increased resistance to env. stress – Drought
• Produce product, other than food– Hormone/enzyme