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RESTRICTION ENDONUCLEASES
• Restriction endonucleases (restriction enzymes): Bacterial
enzymes that cleave double-stranded DNA into smaller, more
manageable fragments
• Each enzyme cleaves DNA at a specific palindromic nucleotide
sequence (4-6bp), producing restriction fragments.
• DNA sequence that is recognized by a restriction enzyme is called
restriction site
• These enzymes form either staggered cuts (sticky or cohesive ends)
or blunt end cuts on the DNA
• Bacterial DNA ligases can anneal two DNA fragments from
different sources if they have been cut by the same restriction
endonucleasethe hybrid combination of two fragments is called a
recombinant DNA molecule
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Genomic DNA libraries:
1.
It is the collection of fragments of double-stranded DNA
obtained by digestion of the total DNA of the organism
with a restriction enzyme.
2.
Ligation of the fragments to a vector.
3.
The recombinant DNA molecules are replicated within
host bacteria.
4.
The amplified DNA fragments represented the entire
genome of the organism and are called a genomic library.
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DNA Sequencing
Cloned
fragment
Primer
Primer Binding sites
Plasmid (or phage)
with cloned DNA
fragment
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The ddATP Reaction
Pol.
3’AATAGCATGGTACTGATCTTACGCTAT5’
Pol.
Pol.
Pol.
5’TTATCG
5’TTATCGTA
5’TTATCGTACCATGA
5’TTATCGTACCATGACTAGA
5’TTATCGTACCATGACTAGATGCGATA
Let me
Through!
Oh come
on!
Not
Again!
Agggg….
5’TTATCGTA
5’TTATCGTACCA
5’TTATCGTACCATGA
5’TTATCGTACCATGACTA
5’TTATCGTACCATGACTAGA
5’TTATCGTACCATGACTAGATGCGA
5’TTATCGTACCATGACTAGATGCGATA
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DNA Sequencing
ddCTP
ddGTP
ddTTP
Read 5’ to 3’ from bottom to top
• Products from 4 reactions each
containing a small amount of a
dideoxynucleotide are loaded onto a
gel
ddATP
• Polyacrlyamide gels capable of
separating fragments differing in size
by only one base
• High concentrations of urea are used
to prevent formation of double
stranded DNA or secondary
structures
• Because polymerization goes 5’ to 3’
shortest fragments are 5’ compared to
longer fragments which are in the 3’
direction
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Synthetic oligonucleotide probes
• If the sequence of all or part of the target DNA is known,
single stranded oligonucleotide probes of 20-30 nucleotides can
be synthesized that are complementary to a small region of the
gene of interest.
• If the sequence of the gene is unknown, the amino acid
sequence of the protein-that is the gene product-may be used to
construct a probe. Short, single-stranded DNA sequences (15-30
nucleotides) are synthesized, using the genetic code as a guide.
• Because of the degeneracy of the genetic code  synthesize
several oligonucleotides.
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• Use of a pair of such ASOs
(one specific for the normal
allele and one specific for the
mutant allele) allows one to
distinguish the DNA from all
three possible genotypeshomozygous normal,
heterozygous, and
homozygous mutant.
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•
Two DNA variations commonly resulting in RFLPs:
1. Single base changes in DNA:
•
About 90% of human genome variation comes in the
form of single nucleotide polymorphisms, or SNPs
(pronounced "snips"), that is, variations that involve just
one base.
• The alteration of one or more nucleotides at a restriction
site can render the site unrecognizable by a particular
restriction endonuclease. A new restriction site can also be
created by the same mechanism.
• In either case, cleavage with an endonuclease results in
fragments of lengths differing from the normal, which can
be detected by DNA hybridization
2. Tandem repeats:
Polymorphism in chromosomal DNA
can arise from the presence of a
variable number of tandem
repeats. These are short sequences
of DNA at scattered locations in
the genome, repeated in tandem
(like freight cars of a train).
• The number of these repeat units
varies from person to person, but is
unique for any given individual and,
therefore, serves as a molecular
fingerprint.
• Cleavage by restriction enzymes
yields fragments that vary in length
depending on how many repeated
segments are contained in the
fragment.
• Variations in the number of tandem
repeats can lead to polymorphism.
Prenatal diagnosis
Families with a history of severe genetic disease, may
wish to determine the presence of the disorder in a
developing fetus by prenatal diagnosis.
Many methods are available but molecular analysis of
fetal DNA promises to provide the most detailed
genetic picture.
Direct diagnosis of sickle cell disease is preformed
using RFLPs
The Sickle Cell Anemia Mutation
Normal b-globin DNA
C
Mutant b-globin DNA
T
T
C
G A
A
G U A
mRNA
mRNA
Normal b-globin
Mutant b-globin
Glu
H2 N
C
C
A T
Val
O
OH
H
CH2
H2C
C OH
O Acid
H2 N
C
C
O
OH
H
CH
CH3
H3C
Neutral
Non-polar
Direct diagnosis of sickle cell disease using RFLPs:
• The genetic disorders of hemoglobin are the most common
genetic diseases in humans.
• In the case of sickle cell disease, the mutation that gives rise to
the disease is actually one and the same as the mutation that
gives rise to the polymorphism. Direct detection by RFLPs of
diseases that result from point mutations is at present limited
to only a few genetic diseases.
• Sickle cell anemia is caused by a point mutation. The sequence
altered by the mutation abolishes the recognition site of the
restriction endonuclease MstII that recognizes the nucleotide
sequence CCTNAGG (where N is any nucleotide).
• Thus, the A to T mutation within a codon of the bs-globin
gene eliminates a cleavage site for the enzyme.
RFLP analysis
• Sickle cell anemia is caused by a point
mutation (A to T mutation (base
substitution) within a codon of the bsglobin gene). The sequence altered by the
mutation abolishes the recognition site
CCTNAGG (where N is any nucleotide).
of the MstII restriction endonuclease
• Normal DNA digested with MstII yields a
1.15 kb fragment, whereas a 1.35 kb
fragment is generated from the ßs gene as a
result of the loss of one MstII cleavage
site.
• Diagnostic techniques for analyzing fetal
DNA provide safe, early detection of
sickle cell anemia, as well as other genetic
diseases.
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PCR
Temperature
100
Melting
94 oC
Extension
Annealing
Primers
50 oC
50
0
94 oC
72 oC
T i m e
3’
3’
5’
5’
3’
3’
5’
5’
5’
3’
5’
5’
5’
3’
5’
5’
5’
3’
5’
5’
5’
30x
Melting
5’
3’
15
3’
5’
3’
Temperature
100
Melting
94 oC
PCR
50
0
T i m e
3’
5’
5’
3’
16
Temperature
100
50
0
3’
5’
5’
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
30x
T i m e
5’
5’
5’
Melting
94 oC
PCR
5’
3’
5’
5’
5’
5’
5’
5’
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Temperature
100
Melting
94 oC
50
0
3’
5’
5’
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
30x
T i m e
5’
5’
5’
PCR
5’
3’
Fragments of
defined length
5’
5’
5’
5’
5’
5’
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DNA Between The Primers Doubles With Each
Thermal Cycle
Number
1
2
0
1
Cycles
4
8
16
32
64
2
3
4
5
6
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Steps of a PCR
• At the completion of one cycle of replication, the reaction mixture
is heated again to denature the DNA strands (of which there are
now four). and the cycle of chain extension is repeated.
• Thus, each newly synthesized polynucleotide can act as a template
for the successive cycles. This leads to an exponential increase in the
amount of target DNA with each cycle, hence, the name
"polymerase chain reaction”
• By using a heat-stable DNA polymerase (for example, Taq
polymerase) from a thermophilic bacterium, the polymerase is not
denatureddoes not have to be added at each successive cycle.
• Each extension product of the primer includes a sequence
complementary to the primer at the 5' end of the target sequence.
• Advantages o PCR: sensitivity (target DNA is less than 1 part in a
106 of the initial sample) and speed ( as compared to recombinant
DNA cloning technology)
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