Genomics & Transcriptome

3(3-0)

BIT-504

Prof. Dr. Roquyya Gul

 ENZYMES FOR
DNA MANIPULATION
 Activities of (A) DnA polymerases, (B) nucleases, (c) ligases, and (D) end -
modification enzymes. (A) Activity of a DNA-dependent DNA polymerase is shown on the left, and
that of an RNA-dependent DNA polymerase is shown on the right. (B) Activities of endonucleases and
exonucleases are shown. (C) Activity of a ligase: the green DNA molecule is ligated to itself (left) or to a
second DNA molecule (right). (D) Activity of terminal deoxynucleotidyl transferase is illustrated: this
enzyme adds nucleotides to the ends of a double-stranded DNA molecule.
 Subdividing the Genome
• Fragmentation of DNA with restriction
enzymes
• Separating large fragments of DNA and to
clone
 Restriction Enzymes
• For example found in bacteria
• Cut DNA within the molecule (endonuclease), cut at ends
(exonucleases)
• Cut at sequences that are specific for each enzyme (restriction
sites)
• Leave either blunt or sticky ends, depending upon the specific
enzyme
 Polymerase Chain Reaction
• PCR allows scientists to amplify small,
specific segments of DNA = make millions
of copies of segment
• Allows for amplification at an exponential
rate
• DNA Replication in a test tube
 Materials needed for PCR
• Target DNA (the DNA you want to copy)
• Free Nucleotides (A, T, C, G)
• Primers
• Taq Polymerase (heat stable DNA Polymerase
III)
• Mg2+ (cofactor that DNA Polymerase III needs
to work)
• Buffer
• Thermocycler (machine that changes
temperatures)
 Overview of PCR
• Uses different temperatures to amplify
DNA
• Step 1: Separate existing DNA strands –
95ºC (Denaturation)
• Step 2: Lower temperature to allow
primers to bind to target DNA – 55ºC
(Annealing)
• Step 3: Raise temperature to allow Taq
Polymerase to build DNA strand – 72ºC
(Extension)
Differences between DNA Replication
& PCR
• No Helicase or Topoisomerase – PCR uses
the first heat step to completely separate the
strands of DNA
• No Primase – primers are already made
• DNA primers – no need for DNA
Polymerase I
• No leading or lagging strands – DNA is
completely unzipped, no Okazaki fragments
 The polymerase chain reaction – used to amplify a
10_27_1_PCR_amplify.jpg
specific DNA sequence with cyclic changes in temperature
Exponential Amplification (at every cycle,
product is doubled)
of template DNA
 Denaturation
• Heating separates the
double stranded DNA
–Denaturation
• Slow cooling anneals Heat Cool
the two strands
–Renaturation
Annealing

• Two primers are supplied in molar excess

• They bind to the complementary region
• As the DNA cools, they wedge between
two template strands
• Optimal temperature varies based on
primer length etc.
• Typical temperature from 40-60 ºC
Extension
• DNA polymerase duplicates DNA
• Optimal temperature 72ºC
• Regions of up to 5kb can be amplified
without much difficulty.
• Longer amplifications; up to 40kb are
possible by modifications of the standard
technique.
• Fragments longer than about 40kb are
unattainable by PCR.
 Gel electrophoresis is used to examine the
results of PCR OR Restriction digestion
• Two types of gel are used in molecular biology:
• Agarose gels and Polyacrylamide gels, which are
mainly used in DNA sequencing.
• Agarose is a polysaccharide that forms gels with pores
ranging from 100 to 300 nm in diameter; the pore size
depends on the concentration of agarose in the gel.
• Gel concentration therefore determines the range of
DNA fragments that can be separated.
• Agarose gel is composed of microscopic pores through
which DNA travels.
• There is an inverse relationship between the pore size
of the gel and the concentration – pore size decreases
as the density of agarose fibers increases.
• High gel concentration improves separation of
smaller DNA molecules, while lowering gel
concentration permits large DNA molecules to be
separated.
• Depending on the concentration of agarose in the gel,
fragments between 100 bp and 50 kilobase pairs (kb)
in length can be separated into sharp bands after
electrophoresis.
• For example; a 0.5 cm thick slab of 0.5% agarose,
have large pores, used for molecules in the size range
1–30 kb.
• 0.3% gel can be used for longer molecules up to 50
kb.
• 5% gel can be used for shorter molecules 100–500
bp in length.
• The bands of DNA can be visualized by soaking the gel in
ethidium bromide solution OR by adding ethidium bromide in
gel.
• Ethidium bromide (mutagenic) intercalates between DNA base
pairs and fluoresces when activated with ultraviolet radiation.
• Non mutagenic dyes that stain DNA green, red, or blue are
now used in many laboratories. The most sensitive dyes are able
to detect bands less than 1 ng of DNA, as compared to a
minimum of 10 ng of DNA when ethidium bromide is used.
 Rate of product formation can be followed during a
PCR
• The synthesis of the DNA product is followed as the PCR
proceeds through its series of cycles is called real-time PCR.
• Carried out in two different ways;
 In the simplest method, a dye that gives a fluorescent signal
binds to DNA is included in the PCR mixture.
 The gradual increase in the fluorescent signal given out by
the mixture indicates the rate at which the product is being
synthesized.
 The disadvantage of this approach is it measures the total
amount of double-stranded DNA in the PCR at any particular
time. This is because the primers sometimes anneal to
themselves in various nonspecific ways, increasing the
amount of double-stranded DNA that is present.
 The second method for real-time PCR requires a short
oligonucleotide called a reporter probe, which gives a
fluorescent signal when it hybridizes to the PCR product.
 The probe hybridizes only to the PCR product, this
method avoids the problems caused by primer–primer
annealing.
 Use of a pair of labels comprising a fluorescent dye and a
compound that quenches the fluorescent signal when
brought into close proximity with the dye.
 This quenching is brought about by a process called Förster
resonance energy transfer (FRET).
 The dye is attached to one end of the reporter probe and
the quenching compound is attached to the other end.
Applications of real time
quantitative PCR
• relative and absolute quantification
of gene expression.
• validation of DNA micorarray
results.
• variation analysis.
• counting bacterial, viral, or fungal
loads.
• to obtain sequences from the trace
amounts of DNA that are present in
hairs, blood stains, and other forensic
specimens and from bones and other
remains preserved at archaeological
sites.
• to amplify DNA from preserved
skeletons has led to genome
sequences of extinct species.
Ligases join DNA fragments together
Figure 2.16 linkers are used to place sticky
ends onto a blunt-ended molecule. In this
example, each linker contains the recognition
sequence for the restriction endonuclease
BamHI. DNA ligase attaches the linkers to the
ends of the blunt-ended molecule in a reaction
that is made relatively efficient because the
linkers are present at a high concentration. The
restriction enzyme is then added to cleave the
linkers and produce the sticky ends. Note that
during the ligation the linkers ligate to one
another, so a series of linkers (a concatemer) is
attached to each end of the blunt molecule.
When the restriction enzyme is added, these
linker concatemers are cut into segments, with
half of the innermost linker left attached to the
DNA molecule. Adaptors are similar to linkers
but each one has one blunt end and one sticky
end. The blunt-ended DNA is therefore given
sticky ends simply by ligating it to the adaptors:
there is no need to carry out the restriction step
 End-modification enzymes
• Terminal deoxynucleotidyl transferase, obtained from calf
thymus tissue an example of an end-modification enzyme.
• It is a template-independent DNA polymerase, because it is
able to extend a DNA polynucleotide without base pairing of
the incoming nucleotides to an existing strand of DNA or
RNA.
• Main role in recombinant DNA technology is in
homopolymer tailing.
• Two end-modification enzymes are frequently used; alkaline
phosphatase and T4 polynucleotide kinase.
• Alkaline phosphatase is obtained from various sources,
including E. coli, calf intestinal tissue, and Arctic shrimp. It
removes phosphate groups from the 5ʹ-ends of DNA
molecules, which prevents these molecules from being
ligated to one another.
• Two ends carrying 5ʹ-phosphates can be ligated to one
another, and an end lacking a phosphate group can be ligated
to an end containing a phosphate, but a link cannot be formed
between a pair of ends if neither carries a 5ʹ-phosphate.
• The use of alkaline phosphatase direct the action of a DNA ligase
so that only desired ligation products are obtained.
• T4 polynucleotide kinase, obtained from E. coli cells infected
with T4 bacteriophage, performs the reverse reaction to
alkaline phosphatase: it adds phosphates to 5ʹ-ends.
• Like alkaline phosphatase, the enzyme is used during complicated
ligation experiments, but its main application is in the end-
labeling of DNA molecules.