BIOLOGI MOLEKULAR DALAM PENELITIAN KEDOKTERAN

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 the various systems of a cell, including the interactions between DNA, RNA and protein biosynthesis as well as learning how these interactions are regulated.

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 enzymatic 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 DNA that codes for a type of protein or for an RNA chain that has a function in the organism.

Difference between RNA & DNA

RNA RNA nucleotides contain ribose sugar RNA has the base uracil presence of a hydroxyl group at the 2' position of the ribose sugar. RNA is usually singlestranded DNA DNA contains deoxyribose DNA has the base thymine Lacks of a hydroxyl group at the 2' position of the ribose sugar. DNA is usually doublestranded

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.

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 genomic DNA (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 in every 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, a molecular biologist who received a Nobel Prize for the discovery 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 for other methods such as sequencing.
A PCR is performed on an automated cycler, which heats and 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.

PCR Analysis

The process follows the principle of DNA replication

17

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 PCRamplified 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 it a new, unique fingerprint.

TECHNIQUES: SOUTHERN BLOT

Southern Blotting (named after Ed Southern, the inventor) is the detection of specific sequences of DNA on a gel by hybridisation with a labelled DNA probe.

TECHNIQUES: SOUTHERN BLOT

Applications:

1) To confirm the presence of a gene, often in conjunction with 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 gene sequence. 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 giving you 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't know 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) protoonco 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 to analyze the expression of thousands of genes simultaneously.

LIMITATIONS OF TECHNIQUES

False positives/negatives Expense Complicated, require high expertise and standardization Cant do them without tissues. Thus clinicians have to collect 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 RNA which is translated to form protein Key processes: DNA replication, transcription, translation

Thanks for your attention

STRUKTUR HIV