Genetic analysis

Southern blot analysis

Loss of genetic material from cancer cells is a classic marker for the presence of potential tumour suppressor loci. Southern blot analysis, using polymorphisms occurring within the human genome (RFLPs), identifies genetic alterations in the cancer cells. It involves homo-genization of tissue samples to extract DNA, followed by cleavage of DNA with restriction endonucleases. Digested DNA is size-separated by gel electrophoresis and transferred to filters for hybridization to labelled probes to provide information on sequences of interest. Limitations:

♦ Low sensitivity for detection of altered sequences.

♦ Large amounts of high-quality genomic DNA needed.

♦ Lengthy procedure.

Many of these have been overcome with the use of polymerase chain reaction (PCR) methodologies, but Southern blot analysis remains the standard in many situations.

Table 3.1 Landmarks in molecular oncology

1953

Watson & Crick, molecular structure of DNA.

1972

First recombinant DNA molecule made with ligase (Paul

Berg).

1974

Doxorubicin, anti-cancer drug, receives FDA approval.

1975

DNA sequencing developed by Frederick Sanger.

1975

Southern blot technique developed to identify DNA

fragments.

1976

SRC, the first human oncogene, discovered. (Now over

50 oncogenes are known.)

1976

Genentech,the first biotechnology firm, founded.

1978

Cisplatin and Tamoxifen, anti-cancer drugs, receive FDA

approval.

1978

Restriction fragment length polymorphisms (RFLPs)

discovered.

1979

p53 gene discovered.

1981

First recombinant vaccine for hepatitus B virus in liver

cancer.

1982

Transgenic mouse overexpressing growth hormone.

1985

Polymerase chain reaction (PCR) invented.

1986

RB1 (retinoblastoma gene), the first of over 20 known

tumour suppressor genes, cloned.

1990

Human Genome Project launched.

1991

Expressed sequence tags (ESTs) developed.

1991

First human gene therapy for cancer (melanoma).

1992

Paclitaxel, anti-cancer drug, receives FDA approval.

1993

hMSH2, the first of the hereditary non-polyposis colon

cancer genes, cloned.

1994/1995

BRCA1/BRCA2, inherited breast cancer genes, cloned.

1996

First eukaryote genome sequenced, Saccharomyces

cerevisiae.

1996

Topotecan, anti-cancer drugs, receives FDA approval.

2000 Completion date for sequence of the complete human genome.

2000 Completion date for sequence of the complete human genome.

A huge resource of micro-satellite markers distributed throughout the genome can be used to map and characterize genetic alterations. Primer pairs flanking regions of interest can be used to amplify the region; the size of product reveals information on the genetic change.

Table 3.2 Tools of the trade

Genetic analysis Molecular Cytogenetics

Fluorescence in situ hybridization Interphase cytogenetics Comparative genomic hybridization Southern blot analysis PCR

DNA microchip technology RNA analysis In situ hybridization Northern blot analysis RT-PCR

Gene expression microarrays Gene expression profiling Differential display RT-PCR Serial analysis of gene expression (SAGE) Suppression subtractive hybridization Protein analysis Immunohistochemistry Western blot analysis ELISA assay Enzyme assays

Electrophoretic mobility shift assay DNA footprinting

Analysis of gene function Gene transfer into tissue culture cell models Transgenic mice Knockout mice

DNA sequence analysis—bioinformatics Database homology searching

GeneBank database of DNA sequences

BLAST sequence similarity analysis

Gene identification

Pattern identification e.g.

transcription factors

DNA structure and composition

Sequence assembly and contig management

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