7.2 Applications and skills

7.2.1 Non-coding DNA and DNA replication

  • Coding sequences of DNA make up less than 3% of the total genome.
  • Some non-coding sequences have a function as introns or telomeres, but most non-coding sequences have no known function.

VNTRs are used in DNA profiling

  • Variable number tandem repeats (VNTRs) are a class of non-coding sequences of DNA that have no known biological function.
  • VNTRs are sequences made of repeating units of 15–100 nucleotides.
  • They are ‘variable’ because the number of repeats differs between individuals, at different loci, on different chromosomes.
  • There are many loci that contain known VNTRs. They can be used in DNA profiling.

Figure 7.2.1a –How VNTRs are used in DNA profilingFigure 7.2.1a –How VNTRs are used in DNA profiling

  • In the simplified example shown in Figure 7.2.1a, a gene from two individuals is cut with a restriction enzyme (Eco RI), and run through an electrophoretic gel (right).
  • On the gel, the DNA at A travels further from the origin because it is shorter than the band at B. The difference in length is a result of the difference in the number of repeats of the tandem sequence.

Skill: Analysing DNA profiles

  • On Figure 7.2.1b the DNA of three hypothetical individuals is shown. The red arrows indicate the boundaries of the VNTRs.

Figure 7.2.1b – VNTR profileFigure 7.2.1b – VNTR profile

Source: Griffiths et al. Introduction to Genetic Analysis, 2002)

  1. Print Figure 7.2.1b, and label each of the bands on the gel using an alphanumeric system from A1 to B6.
  2. State how many loci and how many alleles are being examined.
  3. Determine whether individual A could be the father of either individual B or C. Explain your answer.

DNA sequencing requires inhibition of DNA replication

  • DNA sequencing, using the Sanger method, requires DNA to be replicated in the presence of dideoxyribonucleotides, or ddNTPs.
  • ddNTPs lack the hydroxyl group that would normally form a bond to elongate the DNA backbone.

Figure 7.2.1c – ddNTPs (left) and dNTPs (right)Figure 7.2.1c – ddNTPs (left) and dNTPs (right)

  • Different amounts of four ddNTPs (one of each type for A, T, C and G) are added to reaction tubes that contain single stranded DNA and normal deoxynucleotide triphosphates (dNTPs).
  • The tubes are placed in a machine (similar to a PCR machine) and the DNA goes through many cycles of replication.
  • Whenever DNA polymerase tries to add a ddNTP to a growing DNA strand, it will be blocked, and DNA replication will stop.

Figure 7.2.1d – DNA sequencing using ddNTPsFigure 7.2.1d – DNA sequencing using ddNTPs

  • The resulting DNA strands have varying lengths.
  • The strands are run on a gel and the position of the bases is determined by reading along the four lanes of the gel.

Figure 7.2.1e – Sequencing is faster when results are automatedFigure 7.2.1e – Sequencing is faster when results are automated

  • Usually, the ddNTPs are labelled with fluorescent dyes, so the results can be analysed automatically using computers.
  • Peaks in fluorescence indicate the position of each base in the sequence.

Figure 7.2.1f – Frederick SangerFigure 7.2.1f – Fred Sanger
Fred Sanger (1918–2013) won two Nobel Prizes in his lifetime, the first for discovering the structure of insulin and the second for developing a method for DNA sequencing.

Figure 7.2.1g – VNTR profileFigure 7.2.1g – VNTR
A DNA profile of six individuals using VNTRs from different loci.

Language help

  • VNTRs are sometimes called minisatellite DNA, in order to differentiate them from ‘satellite’ DNA, which is made of short sequences of highly repetitive tandem repeats.
  • The DNA sequencing method described here is the dideoxy-chain termination method, or simply, the Sanger method.

TOK

Highly repetitive sequences were once classified as ‘junk DNA’ by scientists, including Nobel Prize winner Francis Crick! To what extent do authority, labels and categories affect how knowledge is prioritised and obtained?

Figure 7.2.1h – X-ray diffractionFigure 7.2.1h – X-ray diffraction

Nature of Science

Careful observation: Rosalind Franklin’s X-ray diffraction data provided crucial evidence that DNA is a double helix. Review the evidence in 2.2.6. Recall that Franklin did NOT win a Nobel Prize.

Course link

In 3.1.1 you learned that the Human Genome Project was a product of international cooperation and public funding.