Genomics

1
How to characterize a single piece of DNA
- Isolate a small fragment of DNA
- Insert DNA into plasmid (or phage vector)

• Transform recombinant DNA molecule into bacteria

• Amplify DNA by culturing transformed bacteria

-Select for transformants
-Use transformants for variety of purposes
(e.g.expression studies, sequencing, mutational
analysis, etc.)
2
LE 20-2
Cell containing gene of interest
Bacterium
Gene inserted into plasmid
Bacterial chromosome
Plasmid
Gene of interest
Recombinant DNA (plasmid)
DNA of chromosome
Plasmid put into bacterial cell
Recombinant bacterium
Host cell grown in culture to form a clone of
cells containing the cloned gene of interest
Protein expressed by gene of interest
Gene of interest
Copies of gene
Protein harvested
Basic research and various applications
Basic research on gene
Basic research on protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic
waste
Protein dissolves blood clots in heart attack
therapy
Human growth hor- mone treats stunted growth
3
Restriction Enzymes Used to Make Recombinant DNA

• Bacterial restriction enzymes
• cut DNA molecules at specific DNA sequences
  called restriction sites

4
LE 20-3
Restriction site
MemorizeEcoRI restriction site
5
3
DNA
3
5
EcoRI
Restriction enzyme cuts the sugar-phosphate backbo
nes at each arrow.
palindrome
Sticky end
DNA fragment from another source is added. Base
pairing of sticky ends produces various
combinations.
Fragment from different DNA molecule cut by
the same restriction enzyme
Catalyzes phosphodiester bond between 5
phosphate 3 hydroxyl group of sugar
Ligation
One possible combination
DNA ligase seals the strands.
Recombinant DNA molecule
5
Do restriction digests and ligations always work?
What are the other possible undesirable outcomes?
6
What is a common strategy to select for
transformed bacteria?
Grow bacteria on antibiotic only plasmid
carriers will survive
Clever way to select for recombinant clones
Plasmid contains LacZ gene--gt?-galactosidase
X-gal (substrateone product is blue)
Blue colonies
Restriction site in LacZ gene if insert DNA
fragment
?-galactosidase
X-gal
White colonies
7
LE 20-4_3
lacZ gene (lactose breakdown)
Bacterial cell
Human cell
Isolate plasmid DNA and human DNA.
Restriction site
ampR gene (ampicillin resistance)
Bacterial plasmid
Gene of interest
Sticky ends
Human DNA fragments
Cut both DNA samples with the same restriction
enzyme.
Mix the DNAs they join by base pairing. The
products are recombinant plasmids and many
nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells that have
a mutation in their own lacZ gene.
Recombinant bacteria
Plate the bacteria on agar containing ampicillin
and X-gal. Incubate until colonies grow.
Colony carrying recombinant plasmid
with disrupted lacZ gene
Colony carrying non- recombinant plasmid with
intact lacZ gene
Bacterial clone
8
Different goals in creating recombinant clones

• To examine/utilize the structure and function of
  a single
• piece of DNA.

2. To package small pieces of an entire genome
genomic DNA library To have available all the
sequences in the genome for examination and use.
9
LE 20-6
DNA libraries created using plasmids and phage
and bacterial hosts
Foreign genome cut up with restriction enzyme
or
Bacterial clones
Recombinant plasmids
Phage clones
Recombinant phage DNA
Plasmid library
Phage library
Note practical limit on the size of DNA cloned
into a vectors (plasmid 5-10 kbp, phage 45 kbp)
10
How to distinguish one DNA molecule from another?
Characterization of DNA by Size
Agarose Gel Electrophoresis
Digest DNA with restriction enzymes
Load DNA into wells of agarose gel
Apply electric current to fractionate DNA
fragments by size
In electric field with positive and negative
poles, which pole will DNA be attracted to? Why?
11
LE 20-8
Mixture of DNA molecules of differ- ent sizes
Longer molecules
Cathode
Shorter molecules
Power source
Gel
Glass plates
-DNA stained with fluorescent dye (ethidium
bromide) -DNA fluoresces upon exposure
to ultraviolet (UV) light
Anode
12
How would you determine whether a particular gene
or DNA sequence is present in your cloned DNA?
Southern Blot
13
LE 20-10
Heavy weight
Restriction fragments
DNA restriction enzyme
Nitrocellulose paper (blot)
I
?I
??I
Gel
Sponge
Paper towels
I Normal ?-globin allele
I? Sickle-cell allele
I?? Heterozygote
Alkaline solution
Preparation of restriction fragments.
Gel electrophoresis.
Blotting.
Labeled nucleic acid probe RNA or DNA
Southern Blot Analysis
Probe hydrogen- bonds to fragments containing
normal or mutant ?-globin
I
?I
??I
Radioactively labeled probe for ?-globin gene is
added to solution in a plastic bag
I
?I
??I
Fragment from sickle-cell ?-globin allele
Film over paper blot
Fragment from normal ?-globin allele
Paper blot
Hybridization with radioactive probe.
Autoradiography.
14
Why are globin DNA fragments different in size?
15
LE 20-9
Normal b-globin allele
175 bp
201 bp
Large fragment
Ddel
Ddel
Ddel
Ddel
Sickle-cell mutant b-globin allele
376 bp
Large fragment
Ddel
Ddel
Ddel
Ddel restriction sites in normal and sickle-cell
alleles of ?-globin gene
Normal allele
Sickle-cell allele
Large fragment
376 bp
201 bp
175 bp
Electrophoresis of restriction fragments from
normal and sickle-cell alleles
16
Restriction Fragments Length Polymorphisms (RFLP)
- useful in detecting disease alleles
-forensics to identify individuals no two
individuals are alike (exception?)
17
LE 20-17
Defendants blood (D)
Blood from defendants clothes
Victims blood (V)
Do the RFLPs suggest the defendant was in
contact with the victim?
By themselves, do RFLPS prove shes guilty of
assault?
18
Genomics and Molecular Techniques
Ch 20

• Characterization of entire genomes
• Human Genome Project (HPG)
• ambitious goal to sequence the entire human
  genome (initiated 1990 mostly complete 2003)
• Other genomes also sequenced
• Evolutionary relatedness of key interest
• -gtsequence comparison

19
LE 20-11
Chromosome bands
Cytogenetic map
Steps in genome mapping
(chromosome map)
Genes located by FISH
Genetic (linkage) mapping
Genetic markers
Physical mapping
Overlapping fragments
DNA sequencing
20
DNA Sequencing

• Short DNA fragments sequenced by dideoxy
  chain-termination method

21
LE 20-12
DNA (template strand)
Primer
Deoxyribonucleotides
Dideoxyribonucleotides (fluorescently tagged)
3
5
DNA chain terminators
5
DNA polymerase
3
DNA (template strand)
Labeled strands
3
5
3
Direction of movement of strands
Laser
Detector
22

• Other approach to genome sequencing
• Shotgun method
• Sequence random fragments of DNA
• Computer program orders overlapping fragments
  into single continuous sequence

23
LE 20-13
Cut the DNA from many copies of an entire
chromosome into overlapping frag-ments short
enough for sequencing
Clone the fragments in plasmid or phage vectors
Sequence each fragment
Order the sequences into one overall sequence
with computer software
24
Can we learn important information from the
genome sequence?

• Genome organization
• Gene expression patterns in response to
• - environmental change e.g.
• pollution, global warming
• -Development
• embryogenesis-gt
• senescence
• -Disease/Health

25
Computer Analysis Key Tool
Bioinformatics -analysis and storage of
biological data by computing techniques -key
to management analysis of huge data sets
Example Identification of proteins coding
sequences (ORF) in genomes
agatactagcagctctttcgagcatcagcatcaccgatgcatcgatcacg
cgctgtttg
26
Think of a sequence feature that a program could
search for to identify ORFs.
27
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