Molecular Techniques in Biology Flashcards

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1
Q

describe PCR

A
  • heating to 95 degrees causes the H bonds between base pairs to break, forming single-stranded DNA
  • cooling to 55 degrees allows the forward and reverse primers to anneal to the complementary sequences on the 3’ end of the single-stranded DNA through the formation of hydrogen bonds
  • heating to 72 degrees allows for primer extension and is the optimum temperature for Taq polymerase
  • Taq polymerase binds to the 3’ end of primers and adds deoxyribonucleotides that are complementary to the DNA template and catalyses the formation of phosphodiester bonds beween adjacent deoxyribonucleotides
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2
Q

explain the use of PCR

A
  • amplifies specific sequence of DNA within a relatively short time
  • with sufficient amount of DNA, tests for detection of bacteria can be carried out
  • the amount of DNA amplified within a specific duration can allow for determination of viral concentration
  • can be used in the analysis of human DNA e.g. in DNA fingerpringing for paternity testing/criminal investigation
  • can clone genes which can then be used in genetic manipulation
  • can detect genetic defects in early stages/mutations that cause a disease or condition
  • used to amplify and study ancient DNA fragments from fossils
  • can be used to classify organisms by comparing similarities in DNA sequences of different organisms to verify evolutionary relationship
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3
Q

explain the advantages of PCR

A
  • produces large numbers of copies of DNA for analysis in a relatively short span of time
  • cell-free method of DNA replication hence requires no cleanup of unwanted cellular debris/vector DNA
  • able to efficiently amplify targets up to 35kb in length with high fidelity and high yield
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4
Q

explain the limitations of PCR

A
  • need to first know the nucleotide sequence of one short DNA sequence flanking in order to synthesise the PCR primers
  • highly sensitive, could amplify sequences that are not the target sequence if the sample is contaminated with other DNA molecules with sequences complementary to the primers used
  • amplified DNA copies might have changes in their nucleotide sequences due to errors that occur at low but significant frequencies
  • Taq polymerase does not contain a built-in proofreading activity hence it produces a higher than normal frequency of replication errors
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5
Q

briefly describe the principles of gel electrophoresis

A
  • electrical current applied across gel causes negatively charged DNA to move to positively-charged electrode
  • agarose gel acts as molecular sieve for DNA fragment
  • smaller fragments move faster and hence will travel further in a fixed period of time than larger fragments
  • resulting in separation of fragments by size/length/number of electrodes
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6
Q

describe what happens during nucleic acid hybridisation

A
  • at high temperatures, hydrogen bonds are broken between complementary bases, separating double-stranded DNA
  • lowering temperature allows annealing of DNA probes to single-stranded DNA to form hybrid double-stranded DNA
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7
Q

describe gel electrophoresis

A
  • DNA fragments are placed in wells at the negative electrode end of an agarose gel
  • DNA is negatively charged and moves towards the positive electrode when an electric field is applied
  • agarose gel acts as a molecular sieve for DNA fragments
  • distance travelled in a given time depends on the molecular weight of the DNA/larger DNA fragments travel slower and smaller fragments travel faster
  • as DNA is invisible to the naked eye, a small amount of loading dye is added to give an indication of the distance that DNA bands had travelled
  • DNA ladder is used to estimate the size of DNA fragment in the separate bands
  • ethidium bromide can be added to visualise all the fragments under UV light
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8
Q

describe southern blotting

A
  • DNA fragments are transferred from gel to nitrocellulose membrane by capillary action
  • radioactively-labelled DNA probes are added to the membrane which binds to complementary DNA fragments
  • by autoradiography, the positions of bound probes are visualised using photographic film
  • giving rise to dark bands on a light background
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9
Q

explain the use of gel electrophoresis and southern blotting

A
  • allows identification of different alleles/mutations of genes
  • allows DNA fingerprinting
  • allows sequencing of DNA
  • allows purification/isolation of target DNA
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