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
