biomol.ind edit
TRANSCRIPT
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BIOLOGI MOLEKULARDALAM PENELITIAN
KEDOKTERAN
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definition
Molecular biology is the study of molecularunderpinnings of the process of replication,transcription and translation of the genetic
material.Molecular biology chiefly concerns itself with
understanding the interactions between thevarious systems of a cell, including the
interactions between DNA, RNA and proteinbiosynthesis as well as learning how theseinteractions are regulated.
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Since the late 1950s and early 1960s,
molecular biologists have learned to
characterize, isolate, and manipulate the
molecular components of cells and organisms
includes DNA, the repository of genetic
information; RNA, a close relative of DNA; and
proteins, the major structural and enzymatictype of molecule in cells.
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Components involve in molecular
biology
DNA
RNA
Protein
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Gene : Unit of heredity
The DNA segments that carries
genetic information are called
genes.
It is normally a stretch of DNA
that codes for a type of protein or
for an RNA chain that has a
function in the organism.
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RNA DNA
RNA nucleotides contain
ribose sugar
DNA contains deoxyribose
RNA has the base uracil DNA has the base thyminepresence of a hydroxyl group
at the 2' position of the ribose
sugar.
Lacks of a hydroxyl group at
the 2' position of the ribose
sugar.
RNA is usually single-stranded DNA is usually double-stranded
Difference between RNA & DNA
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Protein
Proteins(also known as polypeptides) are
made of amino acids arranged in a linear
chain and folded into a globular form.
The sequence of amino acids in a protein is
defined by the sequence of a gene, which is
encoded in the genetic code.
genetic code specifies 20 standard amino
acids.
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Basic players in molecular biology: DNA, RNA, and
proteins. What they do is this :
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Common sources of DNA :
blood, bone marrow, and tissue ( biopsies and
tissues removed during surgical resections)
Buccal scrapings and hair roots may also be
used.
In forensic applications, semen and vaginalfluids are common sources of DNA.
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Extraction of DNA and RNA
lysis of cells, removal of proteins and other
cellular components, and purification of the
nucleic acids.
RNA extraction are usually made with water
that has been treated with diethyl
pyrocarbonate (DEPC), which also destroys
RNases.
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Gene expression
All cells in your body have the same genomic DNA(up to a very small mutational error), ie. thesequences of nucleotides within thechromosomes are identical.
Not all of the genes in the genome are beingtranscribed and translated into proteins in everycell.
We say that genes which are transcribed &
translated are expressed in the cells. Gene expression controls distinct identities of
cells via functional protein molecules.
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TOOLS USED IN MOLECULAR BIOLOGY
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Techniques: PCR
PCR was first conceived in 1983 by Kary Mullis, amolecular biologist who received a Nobel Prize for thediscovery 10 years later
A PCR (Polymerase Chain Reaction) is performed in order
to make a large number of copies of a gene. Otherwise,the quantity of DNA is insufficient and cannot be used forother methods such as sequencing.
A PCR is performed on an automated cycler, which heatsand cools the tubes with the reaction mixture in a very
short time.
Performed for 30-40 cycles, in three major steps:1)denaturation, 2)annealing, and 3)extension.
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PCR Analysis
The process follows the principle of DNA replication
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USES OF PCR
Genetic Fingerprinting
Paternity testing
Detection of hereditary diseases
Cloning Genes
Mutagenesis
Analysis of ancient DNA
Genotyping of spesific mutation
Comparison of gene expression
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PATERNITY TESTING
Electrophoresis of PCR-amplified DNA fragments.
(1) Father. (2) Child.
(3) Mother.
The child has inherited some,but not all of the fingerprint
of each of its parents, giving ita new, unique fingerprint.
http://en.wikipedia.org/wiki/Image:Pcr_fingerprint.png -
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Techniques: Southern Blot Southern Blotting(named after Ed Southern,
the inventor) is the detection of specific
sequences of DNA on a gel by hybridisation witha labelled DNA probe.
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Techniques: Southern Blot
Applications:
1)To confirm the presence of a gene, often in conjunction withPCR.
2)To test for the presence of a specific allele of a gene (i.e.human disease genetics).
3)To estimate gene complexity, before you have the genesequence.
4) To detect Restriction Fragment Length Polymorphism
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Techniques: Southern Blot
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Techniques: Western Blot
Western blot analysis can detect oneprotein in amixture of any number of proteins while giving youinformation about the size of the protein.
Allows investigators to determine with a specificprimary antibody, the relative amounts of the proteinpresent in different samples.
In clinical settings, Western Blotting is routinely usedto confirm serious diagnosis suggested by ELISA suchas HIV seroconversion
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FISH
Fluorescence In-Situ Hybridization is a method used to
identify specific parts of a chromosome.
For example: sequence of a certain gene, but don't knowon which chromosome the gene is located,
SUSPECTED translocation in a chromosome,
particular defect, based on the appearance of certain
chromosomes, etc.
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Four-color FISH detection of the t(9;22) BCR-ABL translocation. (A) A
normal cell has been hybridized with four FISH probes. The blue and red probeshybridize to sequences in chromosome 22. The yellow and green probes hybridize
to sequences in chromosome 9. The der(22) chromosome, which contains the
BCR-ABL fusion, is shown by the adjacent green and red probes. The der(9) chromosome, which contains thereciprocal ABL-BCR fusion, is shown by the adjacent yellow and blue probes (Courtesy of Cancer Genetics,Inc.).
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Chronic myeloid leukemia (CML) is associated with a highly
characteristic molecular abnormality in hematopoietic stem
cells, the BCR-ABL fusion gene.
This fusion arises from a reciprocal translocation,
t(9;22)(q34;q11.2), that involves the breakpoint cluster region
(BCR) gene on chromosome 22 and the Abelson (ABL) proto-onco
gene on chromosome 9.
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FISH
Applications
Diagnosis in clinical and cancer cytogenetics.
Interspecies studies of evolutionary divergence.
Analysis of aberrations in animal models of humandiseases.
Many more applications.
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Techniques: Microarray
DNA microarraysallow researchers to analyzethe expression of thousands of genessimultaneously.
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Limitations of Techniques
False positives/negatives
Expense
Complicated, require high expertise and standardization
Cant do them without tissues. Thus clinicians have to collectand make databases. tissue banking
Ethical issues raised by testing.
Gambar!!!
Slide
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Conclusions
This has been a general introduction to molecularbiology, introducing the key molecules of life:
DNA(the store of genetic information)
RNA
& protein(the function molecules of the cell)
Central Dogma: DNA is transcribedto form RNAwhich is translatedto form protein
Key processes: DNA replication, transcription,translation