METODE GENETIKA

MOLEKULER

Outline
Teknik dasar untuk mengidentifikasi, mengamplifikasi dan
meng-clone gen
Analisis molekuler DNA, RNA dan Protein
Analisis molekuler Gen dan Kromosom

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Teknik dasar untuk mengidentifikasi,

mengamplifikasi dan meng-clone gen
Gene cloning
DNA Rekombinan
Amplifikasi DNA

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Gene Cloning
Gen kloning adalah isolasi dan amplifikasi gen
tertentu.
Sebuah molekul DNA rekombinan adalah molekul DNA
yang dibuat dengan menggabungkan dua atau lebih
molekul DNA yang berbeda..

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DNA amplification
Perbanyakan jumlah salinan dari fragmen DNA tertentu
melalui replikasi segmen asam nukleat.

Amplifikasi Gen In Vivo

A minichromosome carrying the gene of interest is produced
in the test tube.
The recombinant minichromosome is introduced into a host
cell (such as E. coli), and the host cell replicates the
minochromosome.

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Amplification of a Gene In Vitro

Short DNA strands complementary to DNA sequences
on either side of the gene of interest are synthesized.
These short DNA strands are used to initiate the
amplification of the gene by a heat-stable DNA
polymerase in the polymerase chain reaction (PCR).

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Restriction Endonucleases
Restriction endonucleases make site-specific cuts in
DNA.
The nucleotide sequences are called restriction sites.
Restriction endonucleases protect bacteria from foreign
DNA.
Bacteria protect endogenous restriction sites by
methylation.
Restriction enzymes commonly recognize palindromic
sequences.

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Structure of an EcoRI-DNA Complex

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Many Restriction Endonucleases Make

Staggered Cuts

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 When DNA is cleaved with a restriction endonuclease that

makes staggered cuts, all of the resulting restriction
fragments have complementary single-stranded termini.
The complementary single-stranded termini can hydrogen
bond with each other and be joined together by DNA ligase.

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Construction of Recombinant DNA Molecules In Vitro

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Plasmid Vectors
Circular, double-stranded circular DNA molecules
present in bacteria.
Range from 1 kb to over 200 kb.
Replicate autonomously.
Many carry antibiotic-resistance genes, which can be
used as selectable markers.
Many useful cloning vectors were derived from plasmid
pBR322.

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Bacteriophage Vectors
Most bacteriophage cloning vectors have been constructed
from the phage chromosome.
The central one-third (about 15 kb) of the chromosome
contains genes required for lysogeny but not for lytic
growth.
This portion of the chromosome can be excised and
replaced with foreign DNA.
The foreign DNA inserted must be 10-15 kb.

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Cosmid Vectors
Hybrids between plasmids and the phage chromosome.
Replicate autonomously in E. coli.
Can be packaged in vitro into phage heads.
Accept inserts of 35-45 kb.

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Phagemid Vectors
Contain components from phage chromosomes and
plasmids.
Replicate in E. coli as double-stranded plasmids.
Addition of a helper phage causes the phagemid to
switch to the phage mode of replication, resulting in the
packaging of single-stranded DNA into phage heads.

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Eukaryotic and Shuttle Vectors

Because different organisms use different origins of
replication and regulatory signals, different cloning vectors
must be used in different species.
Special cloning vectors can replicate in other prokaryotes
and in eukaryotes.
Shuttle vectors can replicate in E. coli and in another
species.

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Yeast Artificial Chromosomes (YACs)

Genetically engineered yeast minichromosomes.
Accept foreign DNA inserts of 200-500 kb.
Contain a yeast origin of replication, yeast centromere,
two yeast telomeres, a selectable marker, and a
polycloning site.

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The Polymerase Chain Reaction (PCR)

Synthetic nucleotides complementary to known flanking
sequences are used to prime enzymatic amplification of the
sequence of interest.
Three repeated steps
Denaturation of genomic DNA (92-95C)
Annealing of denatured DNA to oligonucleotide primers (5060C)
Replication of the DNA segment between the sites
complementary to the primers (70-72C)
Amplification occurs exponentially; each cycle doubles the
number of molecules of the sequence of interest.

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Taq Polymerase
DNA polymerase from Thermus aquaticus is used for PCR because it is
heat-stable.
Taq polymerase lacks proofreading activity, so errors are introduced
into the amplified DNA at low but significant frequencies.
When high fidelity is required, heat-stable polymerases with proofreading
activity are used (Pfu or Tli).

Taq is amplifies fragments of DNA larger than a few thousand base

pairs inefficiently.
For amplification of long segments of DNA (up to 35 kb), Tf polymerase is used.

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Applications of PCR

Diagnosis of inherited human diseases (e.g., prenatal

diagnosis).
Identification of individuals in forensic cases from small
DNA samples.

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The Molecular Analysis of

DNA, RNA, and Protein
DNA, RNA, or protein molecules can be
separated by gel electrophoresis,
transferred to membranes, and
analyzed by various procedures.

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Analysis of DNAs by Southern Blot Hybridization

DNA molecules can be separated by size by gel
electrophoresis using agarose or acrylamide gels for larger
and small DNA molecules, respectively.
DNA molecules can then be transferred from the gel onto a
nitrocellulose or nylon membrane using a technique called a
Southern blot.
DNA on the membrane can be hybridized with DNA probes.

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Agarose Gel Electrophoresis

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Southern Blot:
Transferring DNA from the Gel to a
Membrane

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Identification of a Specific Fragment by

Southern Blot Hybridization

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Detection of Wild-Type and Mutant Alleles of

the Cystic Fibrosis Gene

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Analysis of RNAs by Northern Blot Hybridizations

The Northern Blot procedure is nearly identical to Southern
blotting, except
RNA is sensitive to degradation by RNases; contamination with
these enzymes must be prevented.
RNA molecules contain extensive secondary structure and must be
kept denatured during electrophoresis.

Northern blots are useful in studies of gene expression.

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Northern Blot Hybridization Data

(RNA from roots, leaves, and flowers of A. thaliana)

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Analysis of RNAs by Reverse TranscriptasePCR (RT-PCR)

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Analysis of Proteins by Western Blot Techniques

Polypeptides are separated by polyacrylamide gel
electrophoresis in the presence of a detergent that
denatures the proteins.
Proteins are transferred from the gel to a nitrocellulose
membrane.
Individual proteins are detected with antibodies.

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Key Points
DNA restriction fragments and other small DNA
molecules can be separated by agarose or acrylamide
gel electrophoresis and transferred to nylon membranes
to produce DNA gel blots called Southern blots.
The DNAs on Southern blots can be hybridized to
labeled DNA probes to detect sequences of interest by
autoradiography.
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Key Points

When RNA molecules are separated by gel

electrophoresis and transferred to membranes for
analysis, the resulting RNA gel blots are called northern
blots.
RNA molecules can be detected and analyzed by
reverse transcriptase-PCR (RT-PCR).

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Key Points
When proteins are transferred from gels to membranes
and detected with antibodies, the products are called
western blots.

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The Molecular Analysis of

Genes and Chromosomes
The sites at which restriction enzymes cleave DNA
molecules can be used to construct physical maps
of the molecules; however, nucleotide sequences
provide the ultimate physical maps of DNA
molecules.

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Mapping Restriction Enzyme Cleavage Sites

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 Restriction maps reflect true physical distances (unlike

genetic maps).
Restriction maps can be combined with other molecular
techniques to construct physical maps of entire
genomes.

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Techniques Necessary for Sequencing DNA

Restriction enzymes to prepare homogenous samples of specific
segments of chromosomes.
Gel electrophoresis procedures able to resolve DNA fragments
differing in length by a single nucleotide.
Gene-cloning techniques allowing preparation of large quantities
of a DNA molecule.
Sanger sequencing Technique is used to determine nucleotide
sequences.

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DNA Sequencing
A population of DNA fragments is generated.
One end is common to all fragments (the 5 end of the sequencing
primer).
The other end terminates at all possible positions (the 3terminus).

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2',3'-Dideoxyribonucleoside Triphosphates

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Automated DNA Sequencing

Fluorescent dyes are used for detection of DNA chains

instead of radioactive isotopes.
Products of all four chain terminator reactions are separated
through a single gel or capillary tube.
Photocells detect fluorescence of the dyes as they pass
through the gel or capillary tube.
Output of the photocell is directly transferred to a computer
for analysis.

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Automated Sanger DNA Sequencing

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