DD3

Describe genetic screening in cancer?

can identify the specific changes to the genome which have resulted in cancer

Can indicate a certain treatment

Can identify what might be future cancer

Define biomarkers in cancer?

Substances, whose production is increased in cancer cells, or by healthy cells in response to cancer

Where may biomarkers be found?

blood or other clinical samples - blood, urine, stool sample, biopsy

How are most cancer diagnoses carried out?

finding a mass or proliferation of cells

Why is it useful to monitor biomarkers?

Detect what type of cancer it is

What mutations, therefore drug targets

Allow early action to be taken

Monitor stage or success of cancer treatment

Define hybridization?

the joining of two complementary strands of nucleic acids

When does hybridization occur?

Below melting temperature

How to detect hybridisation?

Double stranded DNA has low UV absorption

Single stranded DNA has high UV absorption

As temp increases so does absorbance

Define the melting temperature?

the point where half have been denatured

What temperature is DNA fully denatured?

90-95C

Describe Fluorescence In Situ Hybridization (FISH)?

Modern method for looking at gross changes to genes and chromosomes

Why is FISH useful?

No need to isolate DNA from whole cells can be down with a biopsy or blood sample

Describe the method of using FISH?

Double stranded DNA with fluorescent tag is incubated with sample

The probe is denatured and becomes single stranded

Binds to the complementary region on the chromosome

Describe chromosome inversion?

Get portions of the chromosomes swapping over

Describe chromosome translocation?

Get whole sections of different chromosomes swapping over

Describe gene amplification?

• Get more copies of the gene

• More proteins due to that

Describe Philadelphia chromosome?

A fusion gene via translocation

Describe chromosome inversion/fusion?

Two chromosomes fuse together also one of the chromosomes gets flipped round

How can you use distance in FISH probe?

• Create FISH that are close together on the normal chromosome

  • If get inversion the probes end up further away (break away assay)

What do you need to detect FISH?

florescent microscope

Describe Chromogenic In Situ Hybridization (CISH)?

Can dual-stain a sample using probes with different antigens and secondary antibodies with different enzymes, combination of FISH and IHC

Describe the method of CISH?

• Make probe with a antigen complementary to the target

• Then incubate with a primary antibody that recognised the antigen and binds to it

• The incubate with a secondary antibody that recognises the primary

  • has a conjugated enzyme

• Which when a non coloured substrate is added if the target is present enzyme catalyses the generation of a chromogen

What can be used as the enzyme in CISH?

alkaline phosphatase or horseradish peroxidase

What can be used as the metal in CISH?

gold, silver (get black dots)

What does PCR allow for?

DNA fro a selected region of a genome to be amplified by more than a million-fold provided the part of thesequence is already known

What are the ingredients of PCR?

Template (DNA, cDNA)

Primer (oligonucleotides complementary to the 3 prime end)

DNA polymerase (not human)

4 bases

What is stage 1 of PCR?

Denaturation

Breaking the double stranded DNA → H bonds

By increasing temperature above melting point 94C 2 mins

What is stage 2 of PCR?

Annealing → hybridisation

Cooling with the primers 30-65C 30 secs

Primers bind

Temp depends on primer

What is step 3 of PCR?

DNA synthesis

Use thermostable DNA polymerase (taq/pfu)

Polymerase binds to 3 prime ends and moves along synthesising DNA

Why is the denaturation step shorter in cycle 2 of PCR?

Shorter strands of DNA

How many cycles of PCR does it take to have the exact DNA structure?

3

Define Reverse Transcriptase PCR (RT-PCR)?

A technique for amplifying a specific sequence of RNA by initial conversion to its cDNA

Describe the method used for RT-PCR?

Oligo-dT primer binds to the polyA sequence at the end of the mRNA

Reverse transcriptase produces one strand of complementary cDNA

RNA is removed by RNAase

How does the start of PCR and RT-PCR differ?

RT-PCR has only one strand so only one primer binds, generate 2 strands after the first round of PCR the both primers can bind

What is traditionally used to stain PCR products?

ethidium bromide in agarose gel under UV light

What can also be used to detect PCR products?

primers with a fluorescent label or conjugated enzyme can be used

Describe a multiplex reaction?

Multiple products can be detected on a single gel if their sizes are sufficiently different

How can DNA be detected on a gel?

DNA has negative backbone

Molecules move through the gel from the negative to positive electrode

Smaller molecules travel more rapidly

Describe Capillary Gel Electrophoresis (CGE)?

PCR products are separated by size within a capillary

Can be combined with primers that are tagged

Get peaks when DNA detected

Smaller peaks come first as they travel the fastest

Can more than one reaction in the tube

Why is Real time PCR (qPCR) useful?

Real-time PCR enables quantification of the product in real-time

The amount of template started with

What are the components of Taqman (qPCR)?

Fluorescence reporter, non fluorescent quencher, minor groove binder, oligonucleotide sequence complementary to the target between the forward and reverse primers

How does Taqman work?

• Forward and reverse primers, DNA pol & TaqMan® probe are added to the DNA sample

• Following denaturing and annealing of primers and probe, DNA pol synthesizes new DNA

• Reverse strand synthesized normally

• On forward strand, DNA pol encounters the TaqMan® probe

• 5’ nuclease activity degrades the probe, releasing the fluorescent reporter

How does SYBR Green work?

Binds selectively to double stranded DNA → fluorescence when it does

More double stranded DNA more fluorescence

Define cycle threshold?

the point in which you can start detecting this increase in fluorescence

Start with less template takes longer to get to threshold

How can threshold be used in cancer?

Number of templates can indicate mutations or amplification of genes

Can see if there is a threshold shift in cancer cells