AMED3001 Cancer Cell Practical

Practicals 2 & 3

2022
Agenda

• Administration – where to find the necessary material on Canvas

• Cancer Cell Detective Story:

- Introduction

- Background

- Methods

• Useful linkes to online resources

• Q&A
Canvas à AMED3001 à Modules à MODULE 4: Molecular basis of cancer à Week 7 à Cancer Cell Practical
 Introduction
This practical is designed as a “detective story” to identify three different cancer cell lines based on
their unique molecular profiles by utilising common techniques used by medical researchers,
highlighting the unique nature of each cancer cell.

Aims
1. Identifying the presence of human papillomavirus (HPV) strain 18 DNA in the cancer cell genome using
Polymerase Chain Reaction (PCR).
2. Detect and sequence APC using PCR, RFLP and genomic sequencing to identify if a mutation is present.
3. Look at differences in telomeric foci (ultra-bright telomeres) between the cell lines using
immunocytochemistry (ICC).
 Background - Therapeutic Targeting Cancer Hallmarks

Cell 2011 144646-674DOI: (10.1016/j.cell.2011.02.013)

 Background - Therapeutic Targeting Cancer Hallmarks

Understanding the unique

genomic features of each
patient’s cancer can provide
individualised treatment plans.

Targeted cancer therapies

block the growth and spread of
cancer cells by exploiting
molecular targets associated
with the unique vulnerabilities
of cancer cells.

Beyond targeted therapy, identifying unique

molecular characteristics contribute to
cancer biomarkers discovery and screening.

Cell 2011 144646-674DOI: (10.1016/j.cell.2011.02.013)

Background - APC

https://cancer.sanger.ac.uk/cosmic/census-page/APC
 Background – HPV
E6, E7 E6, E7

HPV E6 and E7 proteins are thought to act as

critical factors in cervical carcinogenesis. E6, E7 E6, E7

E6 E6

E6, E7

E7
E6, E7
Oliveria, L.B. et. al. 2018. ‘Human papillomavirus (HPV) 16 E6 oncoprotein targets the Toll-like receptor pathway’. Adapted from: Mesri E.A. et al. 2014. ‘Human Viral Oncogenesis: A Cancer Hallmarks Analysis’.
 Background – Telomerase and ALT

https://cancer.sanger.ac.uk/cosmic/census-page/TERT
 Methods – DNA Extraction
Step 1
A major objective of cancer research is to gain insight into genetic disorders and
alterations, including somatic mutations and rearrangements, as well as viral infections.
Step 2

In order to save time and gain high quality DNA for downstream applications (PCR), we
use a commercial kit to purify DNA from cell line pellets.
Step 3

In general, the procedure involves:

1. Step 1 – expose the DNA within the cells - lysis of the cell membrane and nuclear
Step 4
membrane to release the genomic DNA from the nucleus using proteinase K
2. Step 2 – bind DNA - selective binding of the DNA on a silica-based column, while
proteins, RNA ect. washed through
3. Steps 3 & 4 – wash DNA - from residual contaminates and salts
Step 5
4. Step 5 – elute - pure DNA

Check out this YouTube video to visualise the procedure. Pure DNA
 Methods - PCR
Polymerase chain reaction (PCR) is a method widely used in molecular biology to rapidly make millions to
billions of copies of a specific DNA sample.
Amplification is achieved by a series of
three steps:
1. Denaturation - double-stranded DNA
templates are heated to separate the
strands
2. Annealing - primers bind to flanking
regions of the target DNA
3. Extension - DNA polymerase extends
the 3ʹ end of each primer along the
template strands.
These steps are repeated (“cycled”) 25–35
times to exponentially produce exact
copies of the target DNA.
Check out this YouTube video (from 1-3.5 minutes) to visualise the procedure.
Taken from: https://www.thermofisher.com/au/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-basics.html
 Methods – DNA Gel Electrophoresis
Gel electrophoresis is a technique used to visualize the DNA fragments.
We will use it to:
1. Visualize DNA fragments after amplification by PCR
2. Separate DNA fragments according to their size after RFLP

DNA samples are loaded into wells (indentations) at one end of a gel, and an electric
current is applied to pull them through the gel.
DNA fragments are negatively charged, so they move towards the positive electrode.
Because all DNA fragments have the same amount of charge per mass, DNA fragments
will be separate according to their size, with small fragments move through the gel faster
than large ones.
By comparing the length to a molecular weight marker (ladder), we can estimate the size
of the amplified products to know if the desired DNA target / region was successfully
amplified.

Check out this YouTube video (first 5.5 minutes) to visualise the procedure.
 Methods – RFLP
Restriction Fragment Length Polymorphism (RFLP) is a technique that uses restriction
enzymes to uncover polymorphisms (changes in the DNA sequence) in DNA sequences.

Restriction enzymes are bacterial endonucleases that cleavage specific DNA sequences.

If a polymorphism occurs in a restriction site, this sequence is no longer identified by the

restriction enzyme and thus not cleaved.

Check out this YouTube video (first 2 minutes) to visualise the procedure.
 Methods – Sanger Sequencing

Sanger sequencing is a method of DNA sequencing

based on the selective incorporation of chain-
terminating dideoxynucleotides (ddATP, ddGTP, ddCTP,
or ddTTP) by DNA polymerase during PCR.
The modern application of this method uses
fluorescently labelled dideoxynucleotides (each
nucleotide with a different fluorophore) with products
separated by size (or length) using capillary
electrophoresis.
The dyes fluorescence are detected and recorded to
output data as fluorescent peak trace chromatograms.

Check out this YouTube video to visualise the procedure.

 Methods – Sanger Sequencing

Sample A Sanger sequencing is a method of DNA sequencing

based on the selective incorporation of chain-
terminating dideoxynucleotides (ddATP, ddGTP, ddCTP,
or ddTTP) by DNA polymerase during PCR.
The modern application of this method uses
Sample C fluorescently labelled dideoxynucleotides (each
nucleotide with a different fluorophore) with products
separated by size (or length) using capillary
electrophoresis.
The dyes fluorescence are detected and recorded to
output data as fluorescent peak trace chromatograms.

Check out this YouTube video to visualise the procedure.

 Methods – BLAST
Basic Local Alignment Search Tool (BLAST) is a bioinformatics tool for sequence searching
(such as DNA sequence).
The query sequence is compared with reference sequences to identify to which sequences it
resembles above a certain threshold.
An excellent and commonly used BLAST was developed and run by the University of
California Santa Cruz: https://genome.ucsc.edu/

For example - the APC gene:

Check out this YouTube video and this one to visualise the procedure.