EXPLORING GENES

Chapter 6
Basic Tools in Gene Exploration

• Restriction Enzymes- molecular scissors

• Blotting techniques-The Southern and
Northern Blots
• DNA sequencing
• Solid phase synthesis of nucleic acids
• The polymerase chain reaction (RT-PCR)
Restriction Enzymes Split DNA
into Specific Fragments
• also called restriction endonucleases
• recognize specific base sequences in
double-helical DNA and cleave at
specific places, both strands of DNA
containing the recognized sequences
• most recognize specific sequences of four
to eight base pairs and hydrolyze a
phosphodiester bond in each strands
• mostly recognize palindromes or inverted
repeats
Restriction enzyme from Streptomyces achromogenes
 Restriction enzymes are used to
cleave DNA molecules into specific
fragments that are more readily
analyzed and manipulated than the
entire parent molecule
Restriction fragments can be separated
by gel electropresis and visualized

• The electrophoretic mobility of a DNA is

inversely proportional to the number of base
pairs.
• Polyacrylamide gels are used to separate
fragments containing about as many as 1000
base pairs
• Agarose gels are used to separate mixtures of
bigger fragments
• Bands can be visualized by autoradiography or
staining with ethidium bromide
Southern Blotting
 DNA is usually sequenced by
controlled termination of replication
(Sanger Dideoxy Method)

• Sanger Dideoxy Methods of DNA

synthesis generates fragments based on
the last base in the sequence
• Based on selective interruption of DNA
synthesis
 DNA Probes and Genes Can Be
Synthesized by Automated Solid-
Phase Methods

• Synthesis of DNA strands is thru the

sequential addition of an ACTIVATED
monomer to a growing chain that is linked
to a solid support
• The activated monomers are the
protonated deoxyribonucleoside-
3’-phosphoramidites
 Uses

• Synthesized oligonucleotide labeled at end

with 32P or a flourescent tag can be used
to search for a complementary sequence in
a very long DNA molecule or even in a
genome consisting of many chromosome
• Synthesis of new tailor made genes
Selected DNA Sequences Can Be
Greatly Amplified by the
Polymerase Chain Reaction
(PCR)

PCR cycle consists of three steps

1. Strands separation
2.Hybridization of the primers
3. DNA synthesis
PCR is a powerful technique in
medical diagnostics, forensics
and molecular evolution
• PCR can detect bacteria and virus with
the use of primers
• PCR is a promising method for the early
detection of cancer
• PCR is use in forensic and legal medicine
because an individual DNA is highly
distinctive
• PCR can amplify DNA that remain intact
for thousands of year so that ancient
DNA can identified and characterized
 Recombinant DNA Technology

• Unrelated genes are combined in the

laboratory
• The resulting recombinant DNA can be
clone by introducing them into a suitable
cells, where they are replicated by the
DNA-synthesizing machinery of the host
Plasmids and Lambda Phage Are
Choice Vectors for DNA Cloning
in Bacteria

• Plasmids are circular duplex DNA

molecules occurring naturally in
some bacteria
 pBR322

• One of the most useful plasmids,

which contains genes for
tetracycline and ampicillin resistance
• Contains restrictions sites for
EcoR1, Sal1 and Pst1
 Lambda (λ) Phage

• Another widely used vector which

can destroy its host or can become
part of it
 M13 Phage

• Another useful vector for cloning DNA

• Useful for sequencing the inserted DNA
• Does not kill its bacterial host
 Specific genes can be cloned
from digests of genomic DNA
 Long stretches of DNA can be
efficiently analyzed by chromosome
walking

• Larger pieces of DNA can inserted

into bacterial artificial chromosomes
(BACS) or yeast aritifical
chromosomes (YACS)

• To scan long regions of DNA,

overlaps in the library fragments are
produced.
 Manipulating the eukaryotic genes

• Eukaryotic genes can be introduced into

bacteria and the bacteria can be used as
factories to produce a desired protein.
• Introduction into animals provides the ability
to examine gene action for gene therapy.
• In regards to plants, inserted genes may
make the plant pest resistant or enables to
grow in harsh condition.
Complementary DNA prepared
from mRNA can be expressed in
host cells
Gene expression levels can be
comprehensively examined
  New genes inserted into eukaryotic
cells can be efficiently expressed

Recombinant DNA molecules can be

introduced into animal cells by:

1. DNA molecules precipitated by calcium

phosphate are taken up by animal cells

2. DNA is microinjected into a cell

3. Viruses are used to bring new genes into

animal cells
Thank
you