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BIOLOGI MOLEKULAR

DALAM PENELITIANKEDOKTERAN



Blood, Vol 86, No 4 (August 151, 1995: pp 1515-1524







DEFINITION

Molecular biology is the study of molecular underpinnings 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.



Since the late 1950s and early 1960s,

molecular biologists have learned to

characterize, isolate, and manipulate the

molecular components of cells andorganisms includes DNA, the repository of

genetic information; RNA, a close relative of

DNA; and proteins, the major structural andenzymatic type of molecule in cells.



COMPONENTS INVOLVE IN MOLECULAR BIOLOGY

DNA

RNA

Protein



GENE : UNIT OF HEREDITY

The DNA segments that

carries genetic information are

called genes.

It is normally a stretch of DNAthat codes for a type of protein

or for an RNA chain that has a

function in the organism.



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



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 aminoacids.



Basic players in molecular biology: DNA, RNA, and

proteins. What they do is this :



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 vaginal

fluids are common sources of DNA.



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.



GENE EXPRESSION

All cells in your body have the same genomicDNA (up to a very small mutational error), ie.the sequences of nucleotides within the

chromosomes are identical. Not all of the genes in the genome are being

transcribed and translated into proteins inevery cell.

We say that genes which are transcribed &translated are expressed in the cells.

Gene expression controls distinct identities of cells via functional protein molecules.



TOOLS USED IN MOLECULAR BIOLOGY



TECHNIQUES: PCR

PCR was first conceived in 1983 by Kary Mullis, amolecular biologist who received a Nobel Prize for the discovery 10 years later

A PCR (Polymerase Chain Reaction) is performed inorder to make a large number of copies of a gene.Otherwise, the quantity of DNA is insufficient andcannot be used for other methods such assequencing.

A PCR is performed on an automated cycler, whichheats and cools the tubes with the reaction mixturein a very short time.

Performed for 30-40 cycles, in three major steps:1)denaturation, 2)annealing, and 3)extension.



17

PCR Analysis

The process follows the principle of DNA replication



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



PATERNITY TESTING

Electrophoresis of PCR-amplified DNA fragments.

(1) Father. (2) Child.

(3) Mother.

The child has inheritedsome, but not all of the

fingerprint of each of itsparents, giving it a new,unique fingerprint.

http://en.wikipedia.org/wiki/Image:Pcr_fingerprint.png



TECHNIQUES: SOUTHERN BLOT Southern Blotting (named after Ed

Southern, the inventor) is the detection of

specific sequences of DNA on a gel byhybridisation with a labelled DNA probe.



TECHNIQUES: SOUTHERN BLOT

Applications:

1) To confirm the presence of a gene, often in conjunctionwith PCR.

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



TECHNIQUES: SOUTHERN BLOT



TECHNIQUES: WESTERN BLOT

Western blot analysis can detect one protein in a

mixture of any number of proteins while givingyou information about the size of the protein.

Allows investigators to determine with a specific

primary antibody, the relative amounts of the

protein present in different samples. In clinical settings, Western Blotting is routinely

used to confirm serious diagnosis suggested by

ELISA such as HIV seroconversion



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'tknow on which chromosome the gene is located,

SUSPECTED translocation in a chromosome,

particular defect, based on the appearance of certain

chromosomes, etc.



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 probes

hybridize 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 the reciprocal ABL-BCR fusion, is shown by the

adjacent yellow and blue probes (Courtesy of Cancer Genetics, Inc.).



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.



FISH

Applications

Diagnosis in clinical and cancer cytogenetics.

Interspecies studies of evolutionary divergence.

Analysis of aberrations in animal models of human diseases.

Many more applications.



TECHNIQUES: MICROARRAY

DNA microarrays allow researchers toanalyze the expression of thousands of genes simultaneously.



LIMITATIONS OF TECHNIQUES

False positives/negatives

Expense

Complicated, require high expertise and standardization

Can’t do them without tissues. Thus clinicians have tocollect and make databases. “tissue banking”

Ethical issues raised by testing.



THANK YOU



Blood, Vol 86, No 4 (August 151, 1995: pp 1515-1524







CONCLUSIONS

This has been a general introduction to molecular biology, introducing the key molecules of life: DNA (the store of genetic information)

RNA

& protein (the function molecules of the cell)

Central Dogma: DNA is transcribed to form RNAwhich is translated to form protein

Key processes: DNA replication, transcription,translation



Thanks for your attention



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