Beruflich Dokumente
Kultur Dokumente
MOLEKULER
Outline
Teknik dasar untuk mengidentifikasi, mengamplifikasi dan
meng-clone gen
Analisis molekuler DNA, RNA dan Protein
Analisis molekuler Gen dan Kromosom
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..
DNA amplification
Perbanyakan jumlah salinan dari fragmen DNA tertentu
melalui replikasi segmen asam nukleat.
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.
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.
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.
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.
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.
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).
Applications of PCR
Southern Blot:
Transferring DNA from the Gel to a
Membrane
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.
John Wiley & Sons, Inc.
Key Points
Key Points
When proteins are transferred from gels to membranes
and detected with antibodies, the products are called
western blots.
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).
2',3'-Dideoxyribonucleoside Triphosphates