as

Francisco J. Hidalgo

Dr. Provost

CHEM 494

March 11, 2015

Real Time Polymerase Chain Reaction

1

Background

● PCR is a technique for amplifying DNA.

● What is needed?

○ DNA template

○ Two primers

○ TAQ polymerase

○ Nucleotides

○ Buffer solution

● The fluorescence of DNA dyes or probes is monitored each cycle

during real time PCR.

2

Background

• PCR is a technique for amplifying DNA.

3

PCR Principle

4

How does Real Time PCR work?

Fluorescent Reporter Probe Method

• To track DNA amplification in real time “DNA-based Probes” are added

• Probe is an oligonucleotide labeled with a fluorescent reporter and a

quencher.

• During the PCR cycle the probe denatures and anneals to the target

sequence

5

How does Real Time PCR work?

• The reporter molecule is only released when a DNA strand is

completely polymerized by TAQ.

6

Output

7

• The point where fluorescence rises above background noise is quantified as the second derivative maximum [crossing point (Cp)] of the curve and correlates to the amount of starting copies within a PCR reaction.

Applications

• Detect nucleic acids that are diagnostic of infectious

diseases, cancer and genetic abnormalities

• Food safety, water quality

• Detection of phytopathogens

• Research

• Others...8

Real-Time PCR Technology for Cancer Diagnostics

• As the number of initial template copies increases, fluorescence appears sooner and the Cp is lower. The relative copy number between two samples (experimental and control) can be determined

by the difference in their Cp values

9

Real-Time PCR Technology for Cancer Diagnostics

● HER-2 and topoisomerase II (topo II)

● The topo II gene is physically located near HER-2, in an area that is

frequently mutated in breast tumors

● DNA amplification of HER-2 can occur concomitantly with topo II

alterations

● Changes in topo II copy number may dictate response to

chemotherapy

10

Real-Time PCR Technology for Cancer Diagnostics

Goal: Determine starting copy numbers of HER-2 and topo II relative to the control gene albumin.

11

as

Francisco J. Hidalgo

Dr. Provost

CHEM 494

March 11, 2015

Real Time Polymerase Chain Reaction

12

Western Blot Technique

Kirsten Monahan

Western Blot Technique

• To separate and identify proteins based on molecular weight

• Protein can be obtained through cell lysis and protein isolation

• Stacking gel: lower pH, low acrylamide concentration so different sized proteins move at same speed

• Separating Gel: high acrylamide concentration so protein bands have better resolution

• Use filter paper to transfer proteins from gel to membrane

• The membrane can be stripped and reprobed with antibodies to search for a different target

What is it used for?

• Gel Electrophoresis

• Results transferred to a membrane band produced for each protein

• Membrane incubated with specific antibodies for the desired protein

• Unbound antibody washed off; antibodies bound to desired protein left and a second antibody incubated on membrane to visualize the first

• Imaged: antibodies detected and only one band should be produced (specific to protein)

• Thickness of band= how much protein present

• A standard loaded indicates amount

●Transfection is  the process of introducing foreign genetic material into a eukaryotic cell.

●A cell that is transiently transfected expresses the foreign gene but does not integrate it into its genome. As a result the new gene will not be replicated.

●These cells express the transiently transfected gene for a finite period of time and afterwards the foreign gene is lost through cell division.

Transient Transfection

By Jilan Knoblauch

Transient transfections are often chemical- or electroporation- based.

Subcutaneous Tumor Formation

“Nude Mice”

•This protocol requires 2–4 hours and presents a method for injecting tumor cells, cancer stem cells or biopsy material into subcutaneous locations within mice.

• The tumor cells or biopsy are mixed with basement membrane matrix proteins (CultrexBME or Matrigel) at 4 °C and then injected into recipient animals at the preferred anatomical sites.

• Tumor cells can also be co-injected with additional cell types, such as fibroblasts, stromal cells, endothelial cells etc…

•Nude mice are acclimated for one week.•One week after implantation of pellets, MCF-7

(human breast adenocarcinoma) tumor cells (107 cells in 100 μL medium mixed with 100 μL Matrixgel™ Basement Membrane Matrix can be injected subcutaneously into the left and right flank respectively.

•Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin in 5% CO2 at 37°C.

• Tumor size and body weights are obtained 2 times per week approximately.

• Tumor weights are calculated from caliper measurements of tumor dimensions in nm suing the ellipsoid formula:

• Tumor volume = 1/2(length × width2)

•length is the longer of the 2 measurements• Generally, tumor size is monitored until an upper weight of 5000mg is attained.

Northern blot

techniques in molecular biology & biochemistry

Rommel Pinlac

Purpose

• to observe gene expression through detection of RNA

• do so in a variety of situations:

• tissues• organs• development stages• environmental stress level• pathogen infection

• in cancer research, used to show overexpression of oncogenes and down regulation of tumor-suppressor genes in tumor cells compared to normal cells

Procedure

Advantages

• detect RNA size• observe alternate splice products• high specificity• step-by-step—can determine whether or

not to blot looking at gel• membrane can be reprobed and used

years after blotting

Out of Date?

• analysis of gene expression normally done by newer techniques

• real time-polymerase chain reaction (RT-PCR)

• Rnase protection assays

• microarrays

• serial analysis of gene expression (SAGE)• huge problem for northern blots is possible RNA degradation by sample

contamination

• another problem is exposure to toxic chemicals (ethidium bromide, formaldehyde, radioactive material, etc.)

Morbidity & Morbundancy

An Educational Production

By Michael Acosta

Morbidity

• “The quality or state of being morbid” (Merriam-Webster).

• “Relating to unpleasant subjects (such as death)/not healthy or normal” (Merriam-Webster)

Morbundancy

• “Guidelines on morbundancy and times for intervention by euthanasia include: impaired ambulation (unable to reach food or water easily, inability to remain upright); evidence of muscle atrophy or other signs of emaciation; any obvious prolonged illness including such signs as lethargy (drowsiness, aversion to activity, lack of physical or mental alertness), prolonged inappetence; difficulty breathing; central nervous system disturbances; or chronic diarrhea or constipation” (IACUC).

Kaplan Meier estimator

• Estimating survival function from lifetime data

Lentiviral Protein Expression

What is it?

• A retrovirus that can infect dividing and non-dividing cells

• Changes the expression of the cells genome for up to six months

• Can be used in gene therapy

•Somatic vs. germ• Commonly used retrovirus is HIV

How does it work?

• Lentiviruses have two strands of RNA

• Ends of virus flanked with long terminal repeats (LTR’s)

•Involved in enhancing, promoting, transcription initiation, transcription termination, and polyadenylation signals• RNA converted to DNA via reverse transcriptase

• Gene products expressed with the help of LTR’s

How are they made?

• Therapeutic gene cloned into a vector sequence flanked by LTRs

• Psi sequence at 5’ end aids in viral packaging

• Viral proteins are not shown in the context of the LTRs

•Not packaged in to virions• Are unable to continue to infect their host after they deliver therapeutic content

Why should you care?

• Can be delivered into any cell

•Ex vivo stem cell stimulation not necessary• No immune responses to lentiviruses have been found

• Can be used to treat a wide variety of diseases – including cancer

Concerns

• That HIV could self replicate and produced via recombination in target cells

• A self replicating infectious vector could cause cancer by activating neighboring proto-oncogenes

Sources

Amado, R. G. and YI. S.. Chen. 1999. Lentiviral Vectors—the Promise of Gene Therapy Within Reach? Science. 285 (5428): 674-76.

Kalpana, G. V. 1999. Retroviral Vectors for Liver-directed Gene Therapy. Seminar in Liver Disease. 19 (1): 27-37

Nichols, E. K. Human Gene Therapy. Cambridge, Massachusettes: Harvard University Press, 1998

HIV as a Vector for Gene Therapy. [Online.] http://bioinformatik.biochemtech.uni-halle.de/uli/genetherapy/genehiv.htm. [12-13-99, last date accessed.]

Thanks!

Any questions?

Invasion & Migration Assay

Biochemistry of Cancer

3-9-15

The Basics

•96-well microplate-based assay•Cell Migration Assay: measures the number of cells traversing a porous membrane

•Cell Invasion Assay: monitors cell movement through extracellular matrices.

Cell Invasion Assay Principle

•Two chambers are separated by a filter coated with extracellular matrix components

•Suspension of cells is placed in the upper chamber and incubated with a media with chemoattractants

•Cells migrate from the top chamber to the bottom through pores

Detection of Invasion

• The assay uses Calcein AM to detect cell invasion.

• Free Calcein fluoresces brightly and is used to quantify the number of cells in the lower chamber in comparison to a standard curve.

Cell Dissociation Solution/ Calcein AM

Fluorescence used to quantify the number of invasive cells

Aspirate both chambers/wash

Incubate

Coated membrane

Bottom Chamber: Test Media

Top Chamber: Cell Suspension

8 μm pore

The Ras/Raf/MAPK/MEK/ERK pathway

Ras

• The initial step in this signaling cascade is a small GTPase (Ras) is activated through a similar interaction that alpha g-proteins undergo when a ligand binds a G-PCR.

• Usually involved with a EGF binding with an EGFR, an assortment of stimulated alpha G-proteins (from GPCR), and tyrosine kinases FYN and FAK.

Raf

• Once Ras phosphorylates Raf, a phosphorylation train begins where Raf phosphorylates MAPK, which phosphorylates MEK and ERK.

• The kinases produced from this pathway have both cytosolic and nuclear targets, changing gene expression that promotes proliferation and inhibits apoptosis.

• This pathway is dysregulated in many types of cancer, but in some types of cancers, it has been indicated that this pathway may arrest the cell cycle.

CRISPRs & Cancer

By: Alex Sidhom

● “clustered regularly interspaced short palindromic repeat”o selective DNA snippets that contain base sequences followed

by short segments of DNA that have previously been exposed to a virus.

● CRISPRs → Cas geneso genomic engineering capabilities

cleave genes or insert new genes● The provision of eukaryotic cells with acquired immune defense

functions that were originally found in prokaryotic cells● Use of an RNA-guided endonuclease to recognize and attach to

foreign, invading DNA at PAM (protospacer adjacent motif)o Dna is degraded/consumed by Cas 9

Introduction

● Use CRISPRS and Cas system to discover and induce combinations of mutations that lead to tumor formationo Recognition of specific genetic mutations that are cancer-

promoting→ use of CRISPRS to cleave those genes

● Creation of cancer modelso identifications of genes “knocked out” in tumors and

metastases discovered suppression of not only tumor-suppressor

genes, but also genes that haven’t previously been linked to cancer

Role in Cancer Therapy

Cell Proliferation Assay

Emily Radke

Methods for Measuring Dividing Cells

• Measure Number/Proportion of Cells Proliferating

• Applications:

•Measure effects of compounds that could either increase or decrease proliferation rates• Four Types:

•DNA Synthesis

•Metabolic Activity

•Antigens Associated with Cell Proliferation

•ATP Concentration

Metabolic Activity

• Dyes that can permeate a cell, interact with enzymes and other factors resulting in a detectable color change as the end product.

• Fluorescence dyes that provide a better sensitivity to the colorimetric dyes.

• Cells are plated and allowed to proliferate, then the dyes are added and a change in color/fluorescence is monitored.

DNA Synthesis

• Reliable and accurate way to measure proliferation by tagging the DNA

• Incubate cells with 3H-thymidine (radioactive) which is incorporated into proliferating cells

•Washed, adhered to filters, counted on a scintillation counter• Substitute BrdU for 3H-thymidine and use antibodies to detect via

fluorescence/colorimeter

Technique: Apoptosis Assays

Jethro Flores

Purpose

• Used to determine selective toxicity of drugs targeted towards cancer cells

Anti-cancer drug

Methods

• Apoptosis in Individual Cells

Apoptosis in Individual Cells

• DNA Fragmentation

•Based on labeling of cellular DNA

•Analyzed by Flow Cytometry and fluorescent/light microscopy• Alteration of permeability and phospholipid composition of plasma membrane

•Permeabilized by ethanol or a detergent

•Light weight molecular DNA leaks out and heavy weight molecular DNA is stained for analysis

Methods

• Apoptosis in Cell Populations

Apoptosis in Cell Populations

• DNA Fragmentation

•Measures pre-lytic fragmentation to determine apoptosis

•Analyzed by DNA ladders and ELISA • Activation of apoptotic proteases (caspases)

•Involved in early stages of apoptosis that triggers a cascade of effects

•Analyzed by in-vitro enzyme assay

•Detection of cleavage of an in-vivo casapase substrate