Lecture 3: Tools of Molecular Pathology Flashcards
Why are the methods used in molecular pathology important?
- understand genes and gene function
- clinical genetics: allows preventative treatments (lifestyle changes to prevent disease
- advancement in treatment of disease: Genotype phenotype relationship, drug function in specific genotype - personalised medicine
What parts of the genome are looked at to understand molecular pathology?
Coding and non-coding regions, regulatory function too
- dna
- rna
- proteins
List the methods for assesing DNA
- PCR and cloning (use gel electrophoresis after)
- hybridisation
- Next-generation sequencing
Generally, what can be detected in DNA assesment
- sequence variation (SNPs and mutations)
- changes in the amount of DNA (Copy Number changes)
Explain what PCR is used for and how it works
- amplifies the small sample of the gene/DNA sample youre interested in
1. template dna is denatured, separating strands
2. Reducing the temperature allows primer to bind to specific spequence of interest
3. Annealing: increase temp, DNA polymerase add nucleotides to DNA, copies DNA, generates double of sample with each pcr cycle
Exponential rise - quick amplification of interested sample
Why is PCR useful
- quick, substitute for cloning, targeted, can detect DNA sequences not normally present (EG.virus), analysis of highly degraded DNA samples (wolly mammoth)
What is Gel Electrophoresis and why is it useful?
mechanism to separate DNA fragments based on size and conformation
- Backbone neg charge, current applied, migrate to positive electrode
- Agorose acts like sieve
- small migrate fast, large migrate slowly
What features are critical to look for when analysing DNA in gel electrophoresis?
- Need positive and negative control for PCR
- Positve will say if it worked (compare size to control)
- Negative (tells if youve contaminated sample, you should never see anything here)
What questions should you ask yourself when anaylsing the DNA sample?
- size of product expected look at + control
- -ve control no band means pcr has worked
- comparison to family
- heterozygous or homozygous (If the two alleles are the same, the individual is homozygous for that particular marker and only one band will appear on the gel. If two different bands appear on the gel, then the individual has inherited two different alleles for that marker and is heterozygous.)
Explain how Sanger sequencing works
- PCR with fluorescent chain terminating ddNTPs
- size separation by capillary gel electrophoresis
- laser excitation and detection by sequencing machine giving chromatogram
How can sequencing be used to assess changes in DNA
- allows for diagnosis by comparison to normal sequence
- see what nucleotides are different
What are the benefits and reprocussions of next generation sequencing
- produce millions of sequences at once
- reduced cost of DNA sequencing
- generation of big data
List the methods for assesing RNA
- RT-PCR
- Next generation sequencing
- Hybridisation
What can be detected in the assesment of RNA?
- read RNA sequence to mind mutations
- count RNA ammount, info about gene expression
- shows DNA translocations make faulty mRNA making bad protein
- mutations in exon splicing
What is RT-PCR and how does it work?
reverse transcriptase polymerase chain reaction
- RNA converted to cDNA by reverse transcription
- cDNA amplified with specific primers and Taq polymerase
Why is real-time RT-PCR useful?
- used to assess the gene expressionor abundance of individual RNAs in a sample
- accumulation of amplified DNA is monitored continously during PCR cycling, via changes in fluoresence
Explain how the change in fluorescence in RT-PCR works
- using TaqMan probes: contain quencher and fluorophore
- bind to gene target in sequence specific manner
- primer elongated by addition of bases, causes fluorofore to be cleaved
- signal when excited by laser
Taq polymerase is the thermostable DNA polymerase while DNA polymerase is the enzyme that catalyzes the synthesis of DNA molecules.
Explain the principle behind a “threshold cycle”
Threshold cylce, fluorescent passes threshold. How many cycles does it take beofre it gves a signal above certain threshold
- Gives info about gene expression
- Low ct: high gene expression, high copy of material (lots to begin with, easily and quickly reaches threshold)
High ct - low gene expression, need lots of pcr cycles beofre you have enough fluorescence to pass threshold
How can Ct cycles be useful in molecular pathology?
ct values can be compared between samples to determine differences in gene expression
What is hybridisation and what is it used for? What types are there?
- hybridisation refers to detecting a specific sequence within a complex mixture of DNA or RNA
- specific detection
- dot blot, southern blot, FISH, SNP, microarrays, aCGH
bold imp ones
What is a microarray?
tool used to detect the expression of thousands of genes at the same time
How do microarrays work?
**1. Sample isolation/preparation **- This step requires the control and target mRNA to be extracted - i.e., a non-cancerous cell line versus a cancerous cell line, respectively. The RNA is then converted into cDNA and labeled with a fluorescent dye
**2. hybridization **- the sample DNA is hybridized with complementary probe sequences on the array chip.
3. washing - Slides are then washed with buffer to remove any DNA that did not strongly hybridize to a probe.
4. image analysis - array chip is exposed to laser excitation showing the relative abundance of hybridized DNA in each target location.
what does the brightness of a spot on the array tell you?
the brighter the spot the more cDNA is present in your sample and the higher its “gene expression”
What is an SNP
A DNA sequence variation that occurs when a single nucleotide in the genome sequence is altered
What function do SNPs provide
serve as landmarks in the search for
- genes associated with disease risk (predisposition to disease)
- drug responses
- complex phenotyes
how do we identify genes associated with disease?
GWAS - genome wide association studies
- search genome for genetic variants (SNPs)
- looks at thousands of nucleotide variants at the same time
case vs control differences
What is aCGH
microarray based technique to detect alterations in genomic DNA sequence
How does aCGH work?
- microarray and hybridisation technique
- looks at chromosomes rather than cDNA
- shows abnormalities, aneuploidies, deletions and duplications
How can proteins be analysed?
- western blot (immunoblotting)
- immunohistochemistry (IHC)
- Immunoassay
What is western blot
- method to identify amount of proteins and their size, including antigens of viruses
- uses antibodies to indetify target proteins among a number of unrelated protein species
What is immunohistochemistry (IHC)?
studies overall gene expression across a slice of tissue to reveal the amount and cellular location of protein but not protein size
how does immunohistochemistry (IHC) work?
uses antibodies to check for certain antigens (markers) in tissue
What is an Immunoassay?
when antibodies are used to quantify the amount of protein or antigen in a lysate
What is the difference between western blotting and Immunihistochemistry
western blotting tells you: size and amount but not cellular locaion
immunohistochemistry: tells you amount and cellular location but not size
