203 L3 Flashcards
Tools of molecular pathology
Molecular pathology is the study and diagnosis of disease through the examination of ——–within organs, tissues or body fluids
Molecular pathology is the study and diagnosis of disease through the examination of molecules within organs, tissues or body fluids
The tools of molecular pathology have played a significant role in:
Understanding genes and gene function
Clinical genetics
- Improved diagnosis (being able to diagnose faster)
- Preventative treatment
Advances in treatment of disease
- Understanding the exact molecular profile of the disorder
- Targeting treatments specifically to the patients disorder
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The human genome is
The set of all genes, regulatory sequences and other information contained within an organism’s DNA
Subdivided into a large nuclear genome and a very small mitochondrial genome
5% of the nuclear genome is highly conserved during evolution = functionally important
Protein coding DNA sequences only account for 1.1% of the genome, the rest = non-coding
mRNA codes for the proteins
Non-coding RNA plays a role in both normal and diseased processes. it is functionally important in the regulation of protein coding genes
Protein-coding genes may belong to families - these arise by gene duplication
Sometimes non-functional gene - related sequence is present = pseudogenes
The non-protein-coding portion of the genome is of crucial functional importance: both for normal development and physiology and for disease
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How can DNA be selectively amplified?
PCR and cloning
What is the end result of PCR and cloning?
Obtain a suffcient and specific quantity of a DNA sequence to be analysed.
Amplification of DNA using PCR
Need to know the specific ——– that you are interested in
Need to be able to design —— that will bind to the ——–
Heating
———– - DNA strands ——– from each other
Cooling
——— - ——– sequences that complement the template of interest bind to the sequences
Heating
——– - DNA ——— adds the bases on
Amplification of DNA using PCR
Need to know the specific region that you are interested in
Need to be able to design primers that will bind to the template
Heating
Denaturation - DNA strands separate from each other
Cooling
Annealing - primer sequences that complement the template of interest bind to the sequences
Heating
Extension - DNA polymerase adds the bases on
Applications of the PCR
A substitute for ——- (chopping up DNA and inserting it into a bacterial plasmid)
Targeted - ——– very specific sequences from small amounts of material
Can selectively detect DNA sequences not normally present in the tissue being tested (e.g. viruses)
Analysis of highly ——— DNA samples
A substitute for cloning (chopping up DNA and inserting it into a bacterial plasmid)
Targeted - amplify very specific sequences from small amounts of material
Can selectively detect DNA sequences not normally present in the tissue being tested (e.g. viruses)
Analysis of highly degraded DNA samples
DNA can be separated by Gel Electrophoresis
Electrophoresis allows separation of a population of DNA according to —— and ———.
DNA is ——– charged so when its put in an electric field it will migrate towards the ——- electrode.
You can tell if there are —— or check that the DNA has been ——.
——- DNA migrate further away from the —— electrode.
Polyacrylamide gels: for single stranded DNA molecules less than 500 nucleotides
Agarose gels: more porous gels for molecules 300-20,000 nucleotides
Pulsed-field gel electrophoresis: for long DNA molecules
Electrophoresis allows separation of a population of DNA according to size and shape.
DNA is negatively charged so when its put in an electric field it will migrate towards the positive electrode.
You can tell if there are deletions or check that the DNA has been amplified.
Smaller DNA migrate further away from the negative electrode.
DNA sequencing
Allows you to ——– nucleotides.
There is a mixture of ——- and —– —-. Anytime there is an incorporation of the ——– base it causes the product to —–.
Each —— base has a different colour detected by the detector. This allows us to reconstruct the ——- of the ——–.
—————– display the sequence of the ——- gene found in the patient compared to the —— gene sequence. Therefore you can use DNA sequencing to find out what the ——— BP were. You cant do this in —— ———.
Allows you to read nucleotides.
There is a mixture of dNTPs and dideoxy dNTP. Anytime there is an incorporation of the dideoxy base it causes the product to stop.
Each dideoxy base has a different colour detected by the detector. This allows us to reconstruct the sequence of the template.
Electropherograms display the sequence of the deleted gene found in the patient compared to the normal gene sequence. Therefore you can use DNA sequencing to find out what the deleted BP were. You cant do this in gel electrophoresis.
Genomic
Uses DNA sequencing and bioinformatics to assemble and understand the structure and function of the genome
First genome was the mitochondrial genome sequenced in 1981
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Improvements in the rate of sequencing due to the improvements in the methods used to sequences
Gel-based systems
Capillary sequencing
Massively parallel sequencing
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Next generation sequencing
Massively parallel DNA sequencing
Producing millions of sequences at once
Reduced cost of DNA sequencing
Massive amount of sequencing data produced = BIG DATA (problem - unintentionally find information that is harmful to the patient)
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RT-PCR (reverse transcriptase-polymerase chain reaction)
examines —— ——-
Often see ————- expression of a specific gene in an ———— place.
Process
Reverse transcriptase phase
—— undergoes a —— synthesis stage to get the —– that can be ———- using the PRC method
Heating
———– - DNA strands ——– from each other
Cooling
——— - ——– sequences that complement the template of interest bind to the sequences
Heating
——– - DNA ——— adds the bases on
examines gene expression
Often see inappropriate expression of a specific gene in an inappropriate place.
Process
Reverse transcriptase phase
RNA undergoes a cdna synthesis stage to get the cdna that can be amplified using the PRC method
Heating
Denaturation - DNA strands separate from each other
Cooling
Annealing - primer sequences that complement the template of interest bind to the sequences
Heating
Extension - DNA polymerase adds the bases on
Real time RT-PCR
Used to assess the gene ———– or ———- of individual RNAs in a sample
Accumulation of amplified —— is monitored continuously during ——– cycling, via changes in ———– signal
Single step amplification and detection
Two ways
Cyber green
Binds to any ——– stranded —- so anytime you get an increase in that product you will get an ——— in this signal.
Sometimes you will get two or more products for example primers binding against each other forming double stranded DNA. Therefore you won’t be able to tell the difference between that double stranded DNA and the double stranded DNA of interest.
TaqMan probes
The Taqman probe binds to gene target in a sequence specific manner.
When the primer pair is elongated by PCR the fluorophore on the probe is cleaved by the Taq polymerase using exonuclease activity.
Displacement away from the Quencher allows the Fluorophore to glow when excited by a laser in the real time PCR machine.
When the probe and the quencher are held close together you don’t end up getting a signal so the fluorescence signal will not release
When the PCR works and there is amplification it causes probe displacement (fluorophore and quencher become separated) causing the release of the fluorescence signal which is detected
So if you have a lot of starting material the fluorescent signal would be amplified compared to if you had less starting material
Used to assess the gene expression or abundance of individual RNAs in a sample
Accumulation of amplified DNA is monitored continuously during PCR cycling, via changes in fluorescence signal
Single step amplification and detection
Two ways
Cyber green
Binds to any double stranded DNA so anytime you get an increase in that product you will get an increase in this signal.
Principal of quantitative real time PCR
In real time PCR we are measuring the ——— ——– which is a specific level of ———- above which you will get an ——— of the product.
Greater the quantity of target ——- (or copy ——-) in the —– material, the ——— a significant ———- in fluorescent signal will appear, yielding a ——— Ct
Ct values can be compared between your samples to determine the gene ——– difference between them
In real time PCR we are measuring the threshold cycle which is a specific level of florescence above which you will get an accumulation of the product.
Greater the quantity of target DNA (or copy DNA) in the starting material, the faster a significant increase in fluorescent signal will appear, yielding a lower Ct
Ct values can be compared between your samples to determine the gene expression difference between them.
