1.1 Blotting

See Table 20.1.

1.2 Polymerase Chain Reaction (PCR)

• aim: amplify specific DNA/RNA sequences in a sample with many DNA/RNA fragments

• if RNA is being amplified, it must first be transcribed into complementary DNA

• steps:

  1. 1. Attach primer onto target DNA

  2. 2. Taq polymerase adds nucleotides onto new DNA strand

  3. 3. Process repeated to produce many DNA copies

1.3 DNA Microarrays

• aim: allows simultaneous analysis of thousands of gene expressions

• microarray is a commercially produced collection of fluorescently labelled DNA short oligonucleotides

• steps:

  1. 1. Isolate RNA from cells

  2. 2. Translate RNA into complementary DNA

  3. 3. Hybridise cDNA onto microarray oligonucleotides

1.4 Bioinformatics

• definition: computing method to store, distribute and analyse large reported DNA information

• researchers report DNA fragment information into databanks

• databanks are accessible via the Internet

1.5 Proteomics

• definition: the study of the structure and function of proteins

2.1 Cancer

• most cancers are caused by mutations in somatic cells (rather than germ cells)

• it takes many years to accumulate the mutations (mutations need at least 20 years to cause cancer)

2.2 Cell Cycle Control Checkpoints

• DNA damage checkpoints:

  1. ◦ where: S phase, G1, G2

  2. ◦ damage detected → cyclin-dependent kinase 2 (CDK2) inhibited → cell cycle progression stopped

  3. ◦ damage not repairable → apoptosis

• spindle checkpoint:

  1. ◦ where: metaphase

  2. ◦ spindle fibres fail to attach to kinetochores → apoptosis

2.3 Signalling Proteins

• signalling proteins include growth factors (+ receptors), signal transduction proteins and transcription factors

• gain-of-function in these proteins can lead to cancer

• mutated alleles usually dominant

2.4 Cell Cycle Control Proteins

• cell cycle control proteins are usually tumour-suppressor proteins

• loss of function in these proteins can lead to cancer

• mutated alleles are usually recessive

• adenomatous polyposis coli (APC):

  1. ◦ function:

     1. activates transcription factor Myc → transcribes genes pushing movement from G1 to S phase

  2. ◦ mutation:

     1. if APC mutated → inappropriately activates Myc → uncontrolled cell proliferation

     2. requires both APC alleles to be mutated for the protein to fail

• p53:

  1. ◦ functions:

     1. detects DNA damage

     2. blocks CDK2

     3. activates apoptosis

  2. ◦ mutation:

     1. requires both p53 alleles to be mutated for the protein to fail

     2. fifty percent of cancers have mutations in p53

• ataxia telangiectasia mutated:

  1. ◦ detects DNA damage

  2. ◦ stops cell cycle

  3. ◦ maintains normal telomere length: prevents chromosome shortening with DNA replication

2.5 Oncogenic Viruses

• some viruses contain proto-oncogenes and oncogenes

• DNA viruses:

  1. ◦ need oncogenes for their own viral survival

  2. ◦ example: human papillomavirus family

• RNA viruses:

  1. ◦ retrovirus enters host → makes DNA copies from viral RNA → DNA copies inserted into host’s DNA for viral replication → oncogenes produced

  2. □ examples:

     1. — Harvey sarcoma virus:

     1. – contains Ha-ras gene (differs from the human ras gene by a single-point mutation)

     2. – Ha-ras overexpression → bladder cancer

  3. — Rous sarcoma virus

     1. – produces v-Src protein, which is a constitutively active mutant of human c-Src protein

     2. – leads to continuous phosphorylation of proteins