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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

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