Molecular Biology Techniques Flashcards

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1
Q

What is the purpose of PCR?

A

amplify specific DNA segment→ sufficient for analysis

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2
Q

What are the components of PCR?

A
  • 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)
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3
Q

What is the role of primers in PCR?

A
  • 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
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4
Q

Explain the advantages of using Taq DNA polymerase

A

Thermostable, won’t denature at high temp→ no need to replace after each cycle→ technique fully automated

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5
Q

Describe the 3 steps in PCR

A
  1. Denaturation: at 95℃: H bonds between cbp of each strand break→ denature dsDNA into ssDNA→ expose bases for cbp
  2. 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
  3. 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
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6
Q

What’s the purpose of a thermocycler and thin-walled tube in PCR?

A

Thermocycler: fully automated

Thin-walled tube: more efficient heat transfer

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7
Q

What are the advantages of PCR?

A
  • 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
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8
Q

What are the limitations of PCR?

A
  1. 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
  2. Need knowledge of seq flanking target region to be amplified→ design primers
  3. Size of DNA to be amplified limited to ~3kb^2→ cannot amplify an entire genome
  4. Minute amounts of contaminant/unwanted DNA are also exponentially amplified to significant amount
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9
Q

Name 3 applications of PCR

A
  • 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
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10
Q

What is the purpose of gel eletrophoresis?

A

separate mixtures of DNA/proteins according to their molecular size→ for analysis & verification of DNA fragments

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11
Q

Outline the principles of gel e

A
  • -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
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12
Q

Describe the protocol of gel e

A
  1. Agarose gel slab placed in buffer solution
  2. DNA sampled mixed with loading dye before being loaded
  3. Load DNA ladder
  4. 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
  5. Current turned off before dye reaches end of gel
  6. Visualisation: gel slab stained w ethidium bromide→ placed under UV light
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13
Q

What is the purpose of a DNA ladder in gel e?

A

This mixture of DNA fragments of known sizes act as a standard to compare w & estimate the size of unknown DNA fragments.

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14
Q

What is the purpose of Southern Blotting?

A

detect and confirm fragments containing specific nt seq are present

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15
Q

Describe the protocol of Southern blotting and explain the significance of each step

A
  1. (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
  2. 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
  3. Wash membrane to remove unhybridised probe
  4. Put X-ray film over the membrane to perform autoradiography to visualise the banding pattern
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16
Q

Explain what is meant by a restriction enzyme

A
  1. Enzyme that recognises & binds to a specific DNA seq called a restriction site, as its AS is complementary to DNA seq
  2. Enzyme breaks phosphodiester bonds on both DNA strands→ blunt or sticky ends ⇒ restriction fragments
  3. 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
17
Q

Explain what is meant by Restriction Fragment Length Polymorphism (RFLP) and how it can be detected.

A
  1. RFLP refers to unique banding pattern among indivs when genomic DNA are digested by RE & after separation by gel e
  2. Due to DNA polymorphism→ variation in no. & location of restriction sites & no. of tandemly repeated nt seq among indivs
  3. To detect: DNA fragments transferred to nitrocellulose membrane and made ss using an alkaline solution
  4. 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
18
Q

Explain how RFLP analysis helped the process of detecting sickle cell anaemia

A
  • 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
19
Q

Explain the banding pattern of individual heterozygous for sickle cell anaemia

A
  • 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
20
Q

Name to applications of RFLP analysis

A

Detection of genetic diseases

DNA fingerprinting in forensics/paternity testing