Chapter 20) DNA Technology and Genomics p. 375-­‐‑383 Old school Biotechnology • Fungi & bacteria to make wine, cheese • Selective breeding New School Biotech • Modify specific species in vitro • recombinant DNA: DNA where the genes are from two different places (usually different species) • genetic engineering: manipulation of genes for practical purposes • biotechnology: manipulation of organisms to make useful products • DNA Cloning o specific genes are hard to find because molecules contain many genes and many nucleotides are non-­‐‑coding – solution = gene cloning o DNA technology makes it possible to clone genes for basic research and commercial applications: an overview  diagram shows how plasmids are used to clone genes  cloned genes may be used to make a protein or make many copies of the gene o
Daniel Oh 2014
Restriction enzymes are used to make recombinant DNA  restriction enzymes: cut DNA at specific locations; protect bacteria against foreign DNA  bacteria cell adds methyl groups to adenines or cytosines to the nucleotides recognized by the restriction enzymes  restriction site: recognition sequence for a restriction enzyme Monday, December 17, 2012 4:41:35 PM Central Standard Time
restriction fragment: series of nucleotides that is made from a restriction enzyme cutting at restriction sites  sticky end: single-­‐‑stranded end of DNA fragments; will form H bonds with complementary base sequences of another DNA molecule cut with the same enzyme (see next page for diagram)  DNA ligase: makes the temporary connection made with H bonds permanent by catalyzing the formation of phosphodiester bonds Genes can be cloned in recombinant DNA vectors: a closer look  cloning vector: original plasmid; DNA molecule that can carry foreign DNA into a cell and replicate  Procedure for Cloning a Eukaryotic Gene in a Bacterial Plasmid • see diagram on 
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Daniel Oh 2014
next page for a way to clone a gene (1) plasmid from E. coli bacterium with two genes and DNA from human tissue cells is isolated (genes are ampR – resistance to amplicillin – and lacZ – encoding the enzyme that catalyzes the hydrolysis of sugar lactose) (2) digest plasmid and human DNA with a restriction enzyme, creating compatible sticky ends on the fragments of both  plasmid and human DNA are mixed together, complementary bases stick together  DNA ligase joins the molecules by covalent bonds (3) bacteria cells take up recombinant plasmids (transformation) (4) transformed bacteria is put on a nutrient medium, where it reproduces (including cloning the human genes) Monday, December 17, 2012 4:41:35 PM Central Standard Time
(5) nucleic acid hybridization: base pairing between gene and a complementary sequence on a nucleic acid molecule; nucleic acid probe: complementary molecule (single stranded, can be RNA or DNA); the probe is traced by labeling it with a radioactive isotope or fluorescent tag (see diagram on next page); denaturation: separation of the two strands of DNA  Cloning and Expressing Eukaryotic Genes: Problems and Solutions • expression vector: cloning vector that has the prokaryotic promoter a little upstream of a restriction site – allow synthesis of eukaryotic proteins in prokaryotic cells • eukaryotic genes have introns – artificial eukaryotic genes are made without introns • mRNA that has had the introns removed can be transcribed back to DNA without introns using reverse transcriptase (see diagram on next page) • complementary DNA (cDNA): DNA without its introns, produced by reverse transcriptase • yeast artificial chromosomes (YACs): have the essentials of a eukaryotic chromosome: origin of replication, centromere, two telomeres • eukaryotic hosts are good to use because many proteins require modifications in order to function, and prokaryotes cannot make these modifications • electroporation: an electric pulse is applied to a solution with the cells – electricity makes a hole in cell’s plasma membrane, so DNA can enter Cloned genes are stored in DNA libraries  genomic library: complete set of recombinant plasmid clones carrying copies of a part of the initial genome  bacteriophages can be used as cloning vectors  cDNA library: represents part of the genome of a cell – used for studying gene functions and tracing gene expression patterns The polymerase chain reaction (PCR) clones DNA entirely in vitro  polymerase chain reaction (PCR): a quicker, more selective method to copy DNA many times (compared to DNA cloning)  only small amounts of DNA is needed for PCR to work correctly  DNA sample does not need to be pure  Involves DNA polymerase from Archae bactium:Thermus aquaticus  Also reqired: deoxynucleoside triphosphates: dATP, dCTP, dGTP, dTTP for making DNA and supplying energy to the replication process  A primer is required. The primer is complimentary to a small sequence of the DNA to be amplified at 3’ end (DNA polymerase works 5’  3’)   process of PCR (see diagram on next page) • double stranded DNA is combined with a heat-­‐‑resistant DNA polymerase and a supply of nucleotides and primers • primers a single-­‐‑stranded DNA complementary to ends of target DNA • at the end of each cycle, the DNA sequence has been doubled • solution is heated again to start another cycle of strand separation, primer binding, and DNA synthesis o
Heat DNA to >95° C o
Cool to 55° C o
Re-­‐‑heat to 72° C and Taq polymerase adds free nucleotides from primers in 5’  3’ direction o
Each cycle lasts about 5 minutes •
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Daniel Oh 2014
Monday, December 17, 2012 4:41:35 PM Central Standard Time
Daniel Oh 2014
Monday, December 17, 2012 4:41:35 PM Central Standard Time
Chapter 20 (Pg. 383-393)
Jeff Yoshimura
DNA Analysis and Genomics
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Uses of duplicated DNA (homogeneous samples)
o Does it differ in different people? Are certain alleles associated with a hereditary disorder? Where
& when is the gene expressed? Location of gene? Differs species to species?
o Need to know complete nucleotide sequence of gene in different people/species to answer
o Genomics – analysis of nucleotide sequences to study sets of genes & their interactions
 Uses gel electrophoresis (fig 20.8, next pg) separates macromolecules (nucleic acids or
proteins) based on their size, electrical charge, physical properties
Restriction fragment analysis detects DNA differences that affect restriction sites
• Detects certain differences in nucleotide sequences of DNA
• Uses gel electrophoresis to sort fragments by size
o Band pattern specific to starting molecule
o Small molecules (bacteria, viruses) can be identified by their band pattern alone
• DNA can be recovered from the gel and can prep. Pure samples of individual fragments
• Different alleles have different patterns; see fig 20.9, next pg
• Starting materials in fig 20.9 are pure & cloned; could have used nucleic acid hybridization instead
o Electrophoresis of whole genome = too many bands to distinguish individually
• Southern blotting (Southern hybridization) – procedure used to compare DNA samples; reveals if
sequence is in DNA sample & the restriction fragments containing the sequence (see fig 20.10)
o Basis for detection is nucleic acid hybridization (last section)
o Possible to compare DNA of different individuals or species
o Starting material can by entire genome (too many bands in electrophoresis for dye, would look
like smear, not bands)
o Radioactively labeled probe selects for bands of interest & exposes them on photographic film
o Sensitivity can be varied to detect only perfect complements to probe or similar seq. as well
o Used non-coding regions to show many differences in band patterns
o Restriction fragment length polymorphisms (RFLPs “rif-lips”) – differences in DNA
sequences on homologous chromosomes  different restriction fragment patterns scattered in
genomes
 Serves as a genetic marker; inherited in Mendelian ways  can make linkage maps
• Frequency with 2 RFLP markers are inherited together ∝ closeness of loci
 Detected & analyzed by Southern blotting
Entire genomes can be mapped at the DNA level
• David Botstein proposes RFLPs could be
map of human genome (1980); now used in
other organisms
• Human Genome Project – (1990) effort to map
entire human genome; sequence nucleotides
o 3 stages: genetic mapping/linkage,
physical mapping, DNA sequencing
o Also maps other species (ex. E. coli,
yeast, fruit fly, etc.)
• Genetic (Linkage) Mapping (stage 1)
o Construct linkage map of 1000s of
genetic markers on chromosomes
o Microsatellites – RFLPs or short
repetitive sequences; enables location
of other markers
 Prepare a probe to match the
3’ end of the known gene
(probe 1)
 Cut the starting DNA with
two restriction enzymes, and
clone the fragments to make 2
libraries
 Use probe 1 to screen library
II for DNA fragments that
overlap the known gene
 Isolate DNA from the tagged
clone, and prepare probe 2 to
match the 3’ end of that
segment
 Use probe 2 to screen library
I for an overlapping fragment
farther along
 Repeat steps 4-5 w/ new
probes to “walk” down DNA
 Result is DNA map w/
known markers in known order
a known distance apart
Figure 20.11 Chromosome walking
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Physical Mapping: Ordering DNA Fragments
o Distances between markers are expressed; usually by number of nucleotides
o Chromosome walking – (fig 20.11) start w/ known DNA segment, use probe to find overlapping
fragments
o First cloning vector: often yeast artificial chromosome (YAC) carries 1 million bp long; bacterial
artificial chromosome (BAC) carries 100,000 – 500,000 bp long
o Determine order of fragments (ex. Chromosome walking) then smaller fragments are made
o Final sets (1,000 bp long) cloned in plasmids or phage; short enough to be sequenced
DNA sequencing (fig 20.12)
o Necessary to be able to clone DNA fragments (key point)
o Start with pure preparation of many copies of short fragment; use sequencing machine
 Combines DNA labeling, DNA synthesis w/ special chain-terminating nucleotides, high
resolution gel electrophoresis
Alternative Approaches to Whole-Genome Sequencing
o J. Craig Venter: skip mapping & just sequence random DNA fragments; use computer to figure
out order based on overlapping short sequences
o Competition between his view & the traditional view  faster processing of human genome
o Certain parts of chromosomes resist mapping (not understood)
(a)
Two homologous segments of DNA that carry different alleles of a
gene are depicted; only the relevant bases are shown. A single basepair difference results in allele 2 having one less recognition sequence
(restriction site) for a particular restriction enzyme. This enzyme cuts
the DNA from allele 1 into three pieces (w,x,& y) but cuts the DNA
from allele 2 into only 2 pieces (z & y)
(b) Electrophoresis separates the restrictyion fragments formed from
each allele. A clear difference between the two alleles is revealed by
their band patterns on the gel. Allele 1 has three bands, corresponding
to w, x, & y; allele 2 has 2 bands corresponding to z & y.
(c) Not pictured. Addition of DNA-binding dye, bands fluoresce pink
in UV light. Yada yada…
Three samples, each containing a mixture of DNA molecules, are
placed in wells near one end of a thin slab of a polymeric gel. The gel is
supported by glass plates and
bathed in an aqueous solution.
Electrodes are attached to both
ends, voltage is applied.
 The DNA molecules, which are
negatively charged, migrate
toward the positive electrode, the
anode. A molecule’s rate of
movement is determined mostly
by its length; longer molecules
travel more slowly through the gel.
 When the current is turned off,
the DNA molecules in each sample
are arrayed in bands along a
“lane”, according to their size. The shortest molecules, having traveled
the farthest, are in bands at the bottom of the gel
Fig 20-8. Gel electrophoresis of macromolecules. Gel electrophoresis
separates macromolecules on the basis of their rate of movement
through a gel in an electric field. For DNA, the migration rate--how far a
molecule travels while the current is on--is inversely proportional to
molecular size. Nucleic acids carry negative charges (on phosphate
groups) proportional to their lengths, but the thicket of polymer fibers in
the gel impedes longer fragments more than it does shorter ones.
Fig 20-9. Using restriction fragment patterns to
distinguish DNA from different alleles.
 Divide DNA frag. Into 4
 Synthesis; randomly a
dideoxynucleotide is inserted
instead of normal one (terminates
growing DNA chain b/c no 3’ end);
this generates random fragments
with a known letter at the end of the
fragment
 Strands separated by
electrophoresis; autoradiography
used to detect radioactive bands
 Sequence read from
autoradiograph
Figure 20.12
Genome sequences provide clues to important biological questions
• DNA technology allows study of genes directly w/o inferring genotype from phenotype (new problem:
what’s the phenotype!)
• Analyzing DNA Sequences
o Use computers to find protein-coding genes (start, stop codons etc.)
o Human Genome found few unique genes (30,000-40,000); much non-coding DNA is repetitive &
long introns
 Alternative splicing & posttranslational processing explain
this
o Human polypeptides more complicated
than invertebrates (domains have more
combinations)
o ½ genes already known; some new ones
similar to old genes (similar protein =
similar duty)
o Similarity in genes  evolutionary
connection between organisms
o Yeast have genes extremely similar to
human ones (can determine human
disease by studying gene in yeast)
o Bacteria have metabolic pathways useful in
medicine/industry
• Studying Gene Expression
o So few genes b/c complex interactions
between them
o Scientists use already sequenced genes to
study patterns of gene expression
Isolate mRNA in cells; make cDNA library by reverse transcription
Compare cDNA with other DNA by hybridization to see which genes are active at diff.
stages of development or in different levels of health
o DNA microarray assays – used to analyze gene expression in an organism
 Probe DNA – short pieces of single stranded DNA attached to glass; sticks to
microarray b/c of positive charge on substrate
 Target DNA – cDNA from cells grown under different conditions
 Compare gene expression in (1) different environmental conditions (dark vs. light; high
vs. low ph); (2) Different times during development (child vs. adult); (3) Different states
of health (pre vs. post-cancer; pre vs. post heart attack); (4) Different body tissues
(muscle types)
  Isolate mRNA;  Make cDNA by rev. transcriptase, using fluorescently labeled
nucleotides;  Hybridization: Apply cDNA to DNA microarray (all wells filled with single
stranded genomic DNA)  check for level of glow (more glow = more expression);
multiple traits checked for with different colors
Determining Gene Function
o Disable gene & look for what is missing
 In vitro mutagenesis – used to cause specific change in sequence of cloned gene;
alter/destroy protein product
 RNA interference (RNAi) – simpler, faster than above; uses synthetic double-stranded
RNA matching a sequence of a particular gene to cause breakdown of mRNA
Future Directions in Genomics
o Proteomics – systematic study of full protein sets encoded by genomes
 Challenging b/c many more proteins than genes; proteins differ with cell’s state & type
 Learn how cells & organisms function
o Bioinformatics – application of computer science * mathematics to genetic & biological info
 Huge amount of data
 DNA variation small in humans b/c new species
 single nucleotide polymorphisms (SNPs) – single base-pair variations in genome
• 99.9% DNA identical between humans
 Need to identify ~3 million SNP sites; used as genetic markers; study evolution
 Identify disease genes; sensitivity to toxins/drugs
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