7.1 - Using gene sequencing

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Last updated 4:27 PM on 8/24/26
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10 Terms

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What is the definition of genome?

The complete set of genetic information contained in the cells of an organism

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What is DNA sequencing?

Identifying the base sequence of a DNA fragment

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How can we amplify DNA fragments in order to sequence them?

Using PCR (Polymerase Chain Reaction), which makes millions of copies of a fragment

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What does the reaction mixture in the first stage of PCR contain?

  • The double-stranded DNA fragment to be amplified

  • Primers that are complementary to each end of the DNA strands

  • Free DNA nucleotides to match up to exposed bases

  • DNA Taq polymerase to create the new DNA


<ul><li><p>The double-stranded DNA fragment to be amplified</p></li></ul><ul><li><p>Primers that are complementary to each end of the DNA strands</p></li><li><p>Free DNA nucleotides to match up to exposed bases</p></li><li><p>DNA Taq polymerase to create the new DNA</p></li></ul><p></p>
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Outline the process of amplifying DNA fragments using PCR

  1. Start with the double-stranded DNA fragment with known end sequences to be amplified

  2. Separate the strands by heating to 95°C to break the hydrogen bonds between complementary base pairs

  3. Add primers that match the end sequences of the DNA strands, then cool to around 55°C - 65°C to allow the primers to anneal (stick) to each end

  4. Heat to 72°C to activate DNA polymerase (extracted from thermostable Taq polymerase) & add free DNA nucleotides

  5. Two new DNA fragments act as templates for the next cycle → repeat the process to obtain many copies of DNA


<ol><li><p>Start with the double-stranded DNA fragment with known end sequences to be amplified</p></li><li><p>Separate the strands by heating to 95°C to break the hydrogen bonds between complementary base pairs</p></li><li><p>Add primers that match the end sequences of the DNA strands, then cool to around 55°C - 65°C to allow the primers to anneal (stick) to each end</p></li><li><p>Heat to 72°C to activate DNA polymerase (extracted from thermostable Taq polymerase) &amp; add free DNA nucleotides</p></li><li><p>Two new DNA fragments act as templates for the next cycle → repeat the process to obtain many copies of DNA</p></li></ol><p></p>
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How can DNA sequencing be used in medicine?

To screen for heritable conditions:

  • when the base sequence of a particular allele or gene is known, we can test a person’s DNA to see if that allele or gene is present


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How can DNA sequencing be used in forensics?

To compare DNA obtained during crime investigations against the DNA of victims or suspects, in order to identify them or discount them

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Aside from forensics & medicine, in which other ways can DNA sequencing be used?

  • To predict the amino acid sequence of proteins

  • To test the relatedness of two individuals (e.g. paternity testing)


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How to calculate the number of DNA fragments formed after PCR?

2n → where ‘n’ is the number of cycles

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Why might PCR not result in the number of DNA fragments that are expected to form?

  • The reaction could run out of primers or nucleotides, meaning DNA replication stops

  • The high temperature of 95°C could damage some of the DNA fragments

  • After many cycles of PCR, even though it is thermostable, DNA Taq polymerase may start to denature