Molecular Biology Techniques Flashcards
What is the purpose of PCR?
amplify specific DNA segment→ sufficient for analysis
What are the components of PCR?
- Template DNA (segment to be amplified)
- Pair of ssDNA primers
- Taq polymerase (catalyse phosphodiester bonds, synthesise DNA strand)
- dNTPs (substrates for DNA replication)
- Buffer containing Mg2+ (cofactor for DNA polymerase)
What is the role of primers in PCR?
- ssDNA that anneal to target DNA seq (complementary)
- Provides free 3’ OH group for chain extension by DNA polymerase
- Complementary to regions flanking gene of interest→ determine segment to be amplified
Explain the advantages of using Taq DNA polymerase
Thermostable, won’t denature at high temp→ no need to replace after each cycle→ technique fully automated
Describe the 3 steps in PCR
- Denaturation: at 95℃: H bonds between cbp of each strand break→ denature dsDNA into ssDNA→ expose bases for cbp
- primer annealing: 55/64℃, w excess primers, 2 primers anneal specifically to regions flanking target DNA/3’ end of template strands via cbp. Primers determine segment to be amplified & provides 3’ OH group for chain extension
- Extension: 72℃, optimum temp for Taq polymerase to elongate chain, via cbp, from 3’ OH end of primer which provides free 3’ OH needed by polymerase→ synthesises complementary DNA strand
What’s the purpose of a thermocycler and thin-walled tube in PCR?
Thermocycler: fully automated
Thin-walled tube: more efficient heat transfer
What are the advantages of PCR?
- Sensitivity: only a minute amount of target DNA needed, as DNA doubles
- Speed: a few hrs needed to amplify quickly and efficiently, compared to cloning which needs at least a week
What are the limitations of PCR?
- Taq pol no 3’ to 5’ proofreading ability→ errors occurring early on gets compounded with each replication cycle & all daughter mlcs from early error are affected
- Need knowledge of seq flanking target region to be amplified→ design primers
- Size of DNA to be amplified limited to ~3kb^2→ cannot amplify an entire genome
- Minute amounts of contaminant/unwanted DNA are also exponentially amplified to significant amount
Name 3 applications of PCR
- Clinical diagnosis: prenatal screening for certain genetic diseases; early detection of viral infections before symptoms appear
- Forensics: identify suspects by amplifying traces of DNA in organic matter
- Study evolutionary relatedness: amplify DNA fragments in prehistoric specimens
What is the purpose of gel eletrophoresis?
separate mixtures of DNA/proteins according to their molecular size→ for analysis & verification of DNA fragments
Outline the principles of gel e
- -vely charged DNA mlcs move toward +ve electrode (anode)
- Meshwork of agarose polysaccharides impedes movement of DNA fragments, impeding longer fragments more than short ones→ longer fragments move slower, end up nearer to well→ separated based on size and hence rate of migration
- Buffer contain ions which allow conduction of electric current→ generate electric field, -vely charged DNA can move from -ve electrode to +ve electrode
- Role of loading buffer (mainly the 1st point)
> Dense loading buffer→ DNA sink to bottom of well located nearest to -ve electrode
> Colours the invisible DNA sample→ show loaded correctly into well
> Allow visualisation of progress of migration
Describe the protocol of gel e
- Agarose gel slab placed in buffer solution
- DNA sampled mixed with loading dye before being loaded
- Load DNA ladder
- Current on→ -vely charged DNA migrates towards the direction of +ve electrode. Meshwork of agarose polysaccharides impedes longer fragments more than short ones→ longer fragments migrate slower, end up nearer to the well→ fragments separate
- Current turned off before dye reaches end of gel
- Visualisation: gel slab stained w ethidium bromide→ placed under UV light
What is the purpose of a DNA ladder in gel e?
This mixture of DNA fragments of known sizes act as a standard to compare w & estimate the size of unknown DNA fragments.
What is the purpose of Southern Blotting?
detect and confirm fragments containing specific nt seq are present
Describe the protocol of Southern blotting and explain the significance of each step
- (Gel slab w DNA placed on a sponge in tray of alkaline solution. On top: nitrocellulose membrane→ paper towels→ heavy weight. Paper towels draw alkaline solution upwards towards themselves, through the gel→)
Alkaline soln denature dsDNA fragments into ssDNA (so probe can bind), which is transferred to nitrocellulose membrane - Nitrocellulose membrane incubated w radioactive ssDNA probe that’s complementary to part of target DNA seq → DNA fragments with target seq hybridise to probe by cbp→ visualisation in later steps
- Wash membrane to remove unhybridised probe
- Put X-ray film over the membrane to perform autoradiography to visualise the banding pattern
Explain what is meant by a restriction enzyme
- Enzyme that recognises & binds to a specific DNA seq called a restriction site, as its AS is complementary to DNA seq
- Enzyme breaks phosphodiester bonds on both DNA strands→ blunt or sticky ends ⇒ restriction fragments
- Used as a defence mechanism by bac against bacteriophage by cutting up foreign DNA into non-infective fragments→ phage cannot replicate→ resistant to phage infection
Explain what is meant by Restriction Fragment Length Polymorphism (RFLP) and how it can be detected.
- RFLP refers to unique banding pattern among indivs when genomic DNA are digested by RE & after separation by gel e
- Due to DNA polymorphism→ variation in no. & location of restriction sites & no. of tandemly repeated nt seq among indivs
- To detect: DNA fragments transferred to nitrocellulose membrane and made ss using an alkaline solution
- nucleic acid hybridization: incubate membrane with radioactive DNA probe, which is complementary to the region of interest & binds to complementary DNA fragments→ appear as bands in autoradiography/X-ray film
Explain how RFLP analysis helped the process of detecting sickle cell anaemia
- Sickle cell anaemia: substitution mutation in DNA coding for ß-globin chain of haemoglobin; at restriction site for Mstll→ Mstll can’t recognises restriction site, does not cleave DNA (‘describe the mutation’)
- Rbc have abnormal, rigid, sickle-shape
- Can infer genotype of indivs using RFLP analysis:
- Genomic DNA from an indiv w sickle cell anaemia & normal indiv are isolated
- Digest w RE, Mstll, then Gel E to separate fragments
- Southern blotting: DNA is made ss using NaOH and transferred to a nitrocellulose membrane, for nucleic acid hybridization using a radioactive probe complementary to β-globin gene
- Visualise via autoradiography. HbS allele→ larger fragment
- A single heavy/larger band→ 2 HbS alleles, a sufferer. 2 bands (one intermediate, one small) → 2 HbA alleles, a normal indiv. 3 bands (one large, one intermediate, one small) → heterozygous, also a normal indiv
Explain the banding pattern of individual heterozygous for sickle cell anaemia
- Heterozygous (3 bands)
- Normal & mutant alleles on the same gene loci
- Normal allele→ intermediate & short fragment when digested by MstII→ correspond to middle and bottommost bands respectively
- Mutant allele→ single long fragment, shown as uppermost band
Name to applications of RFLP analysis
Detection of genetic diseases
DNA fingerprinting in forensics/paternity testing
