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


Watson & Crick, molecular structure of DNA.


First recombinant DNA molecule made with ligase (Paul



Doxorubicin, anti-cancer drug, receives FDA approval.


DNA sequencing developed by Frederick Sanger.


Southern blot technique developed to identify DNA



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

50 oncogenes are known.)


Genentech,the first biotechnology firm, founded.


Cisplatin and Tamoxifen, anti-cancer drugs, receive FDA



Restriction fragment length polymorphisms (RFLPs)



p53 gene discovered.


First recombinant vaccine for hepatitus B virus in liver



Transgenic mouse overexpressing growth hormone.


Polymerase chain reaction (PCR) invented.


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

tumour suppressor genes, cloned.


Human Genome Project launched.


Expressed sequence tags (ESTs) developed.


First human gene therapy for cancer (melanoma).


Paclitaxel, anti-cancer drug, receives FDA approval.


hMSH2, the first of the hereditary non-polyposis colon

cancer genes, cloned.


BRCA1/BRCA2, inherited breast cancer genes, cloned.


First eukaryote genome sequenced, Saccharomyces



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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