DNA Sequencing and Mutation Analysis:

Sanger dideoxynucleotide chain termination procedure: Standard method for small or large scale analysis
Sanger Sequencing DNA is synthesized and all possible length fragments ending with the same nucleotide
1. DNA is cloned into a vector adjacent to primer site denatured in single strands,
primer to template strand, DNA polymerase synthesizes
2. Four ddNTP reactions are carried out A, G, T, C, incorporated to stop synthesis
3. DNA is synthesized, gives rise to sets of DNA products with all possible size
fragments
4. Fragments are analyzed by gel electrophoresis

PCR, multiplex PCR, and their applications to mutation
detection: Important for detecting trinucleotide repeats by
amplifying signal and display products on gel
Lesch-Nyhan syndrome deletions in exons of HGPRT
gene, rapid analysis with multiplex PCR


SNP microarrays or comparative genome hybridization (CGH):
Analysis of entire genome used for high through-put, rapid screening of specific mutations
PCR is used to amplify ligated products
Looking for mutation that prevent hybridization or copy
number loss/increase (CGH)

Copy number variants: Differences in individuals can
lead to disease such as obesity/failure to thrive

Specific mutation detection (ASO or OSH):
Allele-specific oligonucleotide analysis used when mutation is already identified
in patient, test family members,
Check hybridization due to unstable base pair




Next-generation DNA sequencing (NGS), whole-genome,
and whole-exome sequencing:
You usually dont have a large pedigree to determine
inheritance


Gene Manipulation:
Cloning DNA using recombinant DNA techniques:
1. Cut the DNA to separate gene of interest
2. Put DNA in a cloning vector that allows DNA to be replicated
3. Transform bacteria/host cell to mature and produce many copies of
the DNA

Constructing recombinant DNA molecules:
Restriction enzymes bacteria endonucleases used to cut specific symmetrical sequences of 4-8 bp
Ex: EcoR1 cuts each strand at opposite ends to leave overhanging cohesive ends, allows recombination
Vector DNA is delivered to target genome by transformation, Ex: plasmid, bacteriophage, cosmid, yeast
pBR322 plasmid with circular DNA can contains 2 antibiotic resistance genes
Pstl endonuclease cuts DNA of vector and human, human DNA is inserted into vector (transformation), the gene that is
interrupted with human DNA is no longer active

Genomic vs. cDNA clones and how these libraries are constructed:
Genomic clone fragments genomic DNA, exons, introns and promoters
cDNA clone DNA copies of mRNAs, no introns or promoters

Transgenic and targeted mouse models for studying gene function:
Transgenic expression of a gene by random cDNA insertion into genome,
promoter from genomic library is added to manipulate expression
Pronuclear injection linearized genome is injected into male
pronucleus of one cell embryo and planted into uterus, transgene is present in
all offspring DNA inserts randomly and with various copy numbers

Targeted alter gene by homologous recombination with native endogenous gene, done with
mouse embryonic stem cells injected into blastocyst to form chimeric mouse
Knockout deletes endogenous mouse gene or region
Knock-in replaces gene or region with new sequence


Cre/loxP Conditional Knockout/in event occurs during
development in specific tissues, loxP sites on the introns are
hotspots for recombination, Cre recombinase enzyme targets loxP
sites only floxed gene with region knocked out
Tamoxifen activates Cre enzyme gene to express and target loxP
sites for recombination



Therapeutic Complications:
Multidisciplinary approach throughout the process: General physical exam, histopath blood smear, radiology, biopsy
Deliver the news of biopsy results, refer to oncologist, CT staging study, chemotherapy and radiation therapy

Acute toxicities:
GI - Nausea and vomiting, diarrhea, anorexia, constipation, mucositis
Myelosuppression decrease in RBCs, WBCs, thrombocytopenia (platelets)
Marrow toxicity to megakaryocytes, Treat transfusion or prophylaxis
Cardiotoxicity cardiomyopathy (CHF), arrhythmias, tachycardia, ischemia
Pulmonary toxicity pneumonitis, fibrosis, hypersensitivity, pulmonary edema
DTIC Dacarbazine, alkylating agent, hepatic metabolism, IV route, n\v and MYS

Mechanism and treatment of chemotherapy-induced nausea/ vomiting:
Antiemetics to reduce vomiting and dehydration Ondansetron, given before chemotherapy
Acute: occurs on the first day of chemotherapy, interferes with hydration and nutrition
Damage of GI mucosa and stimulation of GI neurotransmitters, activation of chemoreceptor trigger zone in the
brain releases neurotransmitters that induce vomiting (Dopamine, serotonin, histamine)
Delayed: occurs 2 days after perhaps due to residual drug, interferes with hydration and nutrition
Anticipatory: conditioned response in patients with poor emetic control after initial dose or just the thought of it

Neutropenic fever and Treatment: Considered a medical emergency! Infection with fever, lack of neutrophils
Usual infection symptoms are absent, no neutrophils = no inflammatory response
Treatment: Broad spectrum antibiotics given at the site of infection, transfusion

Common long term complications: Infertility, secondary malignancies, pregnancy outcome, myelosuppression


Cancer Biology:
The hallmarks of cancer:
Resist apoptosis, self-sustained growth signals (oncogenes), evading
growth suppressors (tumor suppressor genes), activation of
invasion and metastasis, angiogenesis, replicative immortality

Chemotherapy vs. targeted therapy:
Chemotherapy indiscriminately targets diving cells by blocking DNA
replication and mitosis Antimetabolites, Alkylators, Topoisomerase inhibitors, MT poisons
Targeted therapy affects hormone, immune, and signaling pathways by targeting specific proteins
Antibodies - target specific cell surface antigen to inhibit signaling initiation, used in concert with chemotherapy,
fewer side effects and higher specificity
Small molecules - target oncogene product inside the cell, inhibit key signaling steps, safer than chemo, fewer side
effects and higher specificity

Targeted therapies for cancer-relevant pathways: Technically not "chemotherapy"
RTK inhibitors - Ras/MAPK pathways, VEGFR to prevent angiogenesis
mTOR inhibitors PI3K/AKT/mTOR pathways to prevent cell proliferation, Rapamycin
Target mutated protein, BCR-ABL, BRAF-V600E (melanomas)

Clinical Examples: -mab suffix = monoclonoal antibody
Breast cancer over-expression of Erb-B2 receptor due to gene duplication Ras/MAPK pathway
Treatment: mAb Herceptin to block Erb-B2
Colorectal cancer - deficient MSH2/MLH1 and APC/ -catenin, mutations in K-Ras
Ras/MAPK
Treatment: mAbs Cetuximab and Panitumumab target receptors
Non-small cell lung cancer - mutated EGFR Ras/MAPK
Treatment: TKIs Erlotinib and Gefitinib target mutated receptors inside the cell
Renal cell carcinoma and von Hippel Lindau - VHL mutations inappropriate HIF1-
activation and induction of hypoxia genes: VEGF, TGF, EPO angiogenesis
Treatment: Temsirolimus targets mTOR, Bevacizumab targets VEGF, and Sunitinib
targets growth factor receptors
Chronic Myelogenous Leukemia - BCR-ABL fusion cell growth and apoptosis
Treatment: Imatinib targets BCR-ABL fusion protein
Melanoma - BRAF-V600E mutation Ras/MAPK signaling, Treatment: PLX4032 (Vemurafenib)

**Many targeted therapy drugs target the Ras/MAPK
pathway!**

Emerging hallmarks and enabling characteristics:
Deregulating cellular energetics, avoiding immune
destruction, genome instability and mutation, tumor-
promoting inflammation