DNA Sequencing and Amplification

  • DNA can be sequenced by replicating with a dideoxynucleotide triphosphate – that is a deoxynucleotide triphosphate with no OH group on Carbon 3 of the sugar. This is where a phosphate group would normally bind as part of a DNA backbone – and as this is no longeran option the replication stops when this dideoxynucleotide is added.
    (The small difference between deoxynucleotides and dideoxynucleotides)
  • I’ll try to explain further. If you were looking for all the adenine positions in a DNA strand, you would add ddATP (dideoxyadenine triphosphate) which would cut the replicated strands in different adenine positions.

5′-TCAAGTTACCGTAATA (correct, using dATP)
—- Possible Outcomes —-

This would leave you with a DNA mixture containing different size DNA fragments, all cut after an Adenine residue. To assess the location of these cuts:

  • Denature the dsDNA (double stranded DNA) – this unpairs the new (fragmented) strands from the old DNA strand.
  • Seperate the DNA by polyacrylamide gel electrophoresis (or use agarose gel).
  • Smaller DNA fragments will travel further, faster than larger DNA fragments, and these fragments will be visible after the addition of a florescent chemical.

Now you know the location of Adenine bases, you can repeat the above with ddGTP, ddTTP or ddCTP, revealing the locations of those bases. You could then compare the seperate gels to work out the DNA sequence. To compare, you would need to run on the same gel:

The -ve control would either be clean or just the original DNA strand – to be used as a reference. The +ve would contain a mix of all of the mixtures.

Then it’s a simple case of looking through the different bands. Remember that towards the top are larger fragments and smaller fragments at the bottom – so you read the sequence from the bottom.

CACTCAGTGATG – and the final top strand is the full DNA strand.

– Amplifying DNA – PCR

To amplify a sample of DNA (polymerase chain reaction):

  1. Denature the double stranded DNA sample to leave single stranded DNA (heat).
  2. Add short primers that a complementary to the ends of the sequences of interest.
  3. Lower temperature and anneal.
  4. Use a thermostable DNA polymerase (a polymerase stable under heat) such as Taq polymerase to extend from the primers.
  5. Denature sample again and repeat from 3.

Repeated, this produces exponential amplification of a DNA sample – eg 40 repeats gives 2^40 amplification! This is assuming good conditions with thermostable DNA polymerase and presence of enough dNTP’s (deoxynucleotide triphosphates) and RNA primers.

DNA Replication

  • DNA is copied semi-conservatively. This means that each old strand of DNA pairs with a strand made from new nucleotides.
  • Replication starts at a fixed point and is bidirectional (replicates in both directions). In Eukaryotic DNA, there are multiple replication forks. Eg. E.Coli:
  • The DNA duplex is opened up and nucleotides read 3′-5′ on the OLD strand.. Eg.
  • This means DNA Polymerases synthesise DNA in the 5′-3′ direction.
  • Replication starts from an existing primer. (A primer is a small oglionucleotide sequence that has been made by Primase.)
  • The addition of a nucleotide to the strand involves the removal of 2 phosphate groups from a deoxynucleotide triphosphate as only 1 phosphate is needed for the backbone. This means the addition units (the deoxynucleotide triphosphates) leave behind a 2 phosphate complex known as a pyrophosphate.

The deoxynucleotide is the nucleotide that attaches to the DNA chain below. The deoxynucleotide molecule can also be called a deoxynucleotide monophosphate, for obvious reasons.

I found the above image on the website of the Chemistry & Biochemistry department at the University of Texas at Austin, US. It shows the old strand (blue) unzipping and then new strands binding to this and forming.

Q: “I thought you said it only synthesised in the 5′-3′ direction! How can both new strands be formed especially when the one on the left seems to be going 3′ -5′?”

A: Easy. It still synthesises 5′-3′, it just synthesises in chunks. Hopefully the image below will explain:

As the double DNA strand unzips, the leading prime is free to synthesise a new chain directly in the 5′-3′ direction. That’s how the DNA polymerase works.

But as it can’t synthesise DNA in the 3′-5′ direction it instead synthesises short 5′-3′ fragments for the lagging phase – these are called okazaki fragments and are later joined by DNA ligase.

DNA Structure

DNA is a polymer with a Sugar-Phosphate repeating backbone. Each sugar has a nitrogenous organic base attached (either Adenine, Thymine, Cytosine or Guanine).

Two Deoxyribose Sugars attached by Phosphate Group - DNA Backbone

DNA backbone. You’ve got a deoxyribose sugar attached to a phosphate group & your sugar attached to a base via a BETA-glycosidic bond (eg forms via condensation reaction). Next to look at the bases!

There are two types of bases – Pyrimidine and Purine. Purines are BIGGER with 2 rings (imagine the bigger work being a smaller molecule).

– Pyrimidines (single ring) – T and C & Purines (double ring) – A & G

  • A and T form 2 hydrogen bonds while C and G form 3.
  • The number of H bonds is important as this will determine the strength of the DNA. DNA with a high CG content will have a higher decomposition temperature than DNA with a high AT content because it has a larger number of H bonds. These H bonds hold the helix together better, requiring more energy to break the helix apart.

A little terminology:

Sugar + Base = Nucleoside

Nucleoside + Phosphate = Nucleotide

A deoxynucleoside is a deoxysugar + base.

An oglionucleotide is a polymer of repeating nucleotides in a chain with less than 20 repeat units.

A polynucleotide is a longer chain of repeating units.

– Structure of a DNA Helix

  • Bases are hydrophobic and stack on top of each other in the helix.
  • Phosphates are on the outside – so the helix is highly -vely charged.
  • DNA helix has 2 strands – these run ANTI-PARALLEL. Eg.
  • It’s a right handed helix. Imagine a screw – spirals look like they are going down clockwise.

  • Remember that direction! I’ve drawn an extra white arrow going up on the second picture to show what the 5′—->3′ does.

  • This is also known as B-DNA.

– Other (more rare) helix structures

  • A-DNA. Helix follows same direction but the bases are pulled away from the centre of the helix nearer to the backbone. This happens when the DNA is dehydrated.
  • Z-DNA. A normal helix…but backwards! The DNA strands are wound the opposite way, meaning it appears mirrored to normal B-DNA. This occurs in some GC containing sequences at high salt concentrations.

– Packing down the DNA Helix to more condensed structures (eg. Chromatin)

  • Although the DNA helix is a fairly compact structure, this can be further wound to reduce it’s size. Imagine a helix is like a coil, then winding that coil round and round a roll…this picture borrowed from Wikipedia explains visually.
    (Click picture to enlarge in a new window/tab)
  • Chromatin contains 5 main proteins (H1, H2A, H2B, H3, H4) which are very basic (AKA contain lots of +vely charged amino acids like arginine and lysine) and which interact with the -vely changed DNA (due to the phosphate groups as explained previously).
  • 2x (H2A, H2B, H3) form the round structure you see in the second box of the image above. The DNA helix wraps around this 1.6 times before wrapping round another cylindrical structure.
  • Essentially the DNA is arranged around the histone cores like beads on a string which further packs down to form higher order structures such as chromosomes.

DNA – Introduction to Deoxyribonucleic Acid

Simply, DNA –(transcription)–> RNA –(translation)–> Proteins. (DNA is the template for protein)

Proteins are useful for structure, metabolism, organisation and development…quite essential. What the protein does is dependant on the genes activated and the tissue it is being produced in. A few examples include Keratin which is part of hair (a fibrous protein) and Haemoglobin which is found in red blood cells and binds to Oxygen (a globular protein).

How do we know DNA is the genetic material…and not protein?

In 1928, Frederick Griffith ran experiments with Streptococcus Pneumoniae where he demonstrated how harmless strains could be turned into virulent, harmful strains. He did this by mixing heat killed (therefore protein structure broken) virulent bacteria with live non-virulent forms; resulting in a permanent transformation to a virulant form.

Virulent mixed with non-virulant bacteria

Virulent mixed with non-virulent bacteria

A later discovery in 1944 by Avery was that it was DNA from the virulent cells – not the protein – as DNA is only destroyed by DNase and not by proteases or RNase…which would destroy protein, leaving only DNA.

Later experiments further confirmed this

1. DNA Deoxyribonucleic Acid.

  • Discovered in 1869 in the cell nucleus of Eukaryotes. In Prokaryotes (eg Bacteria) it is not membrane bound and so is ‘loose’ in the cell.
  • Strands of DNA form Chromosomes in Eukaryotes.

– Chromosomes

  • Named Chromosomes because special dyes could pick out AT and CG rich bands – allowing structures to be seen (see Fig 1).
  • Chromosomes are formed from DNA tightly wound round structural proteins such as histone proteins.
  • Cells of a species have the same number of chromosomes. (eg. Humans have 23 pairs in every diploid cell, and 23 in every hapoid cell.)
  • All cells apart from gametes (sex cells) are diploid…aka have a two of each chromosome (so in humans, 46 total). Gametes are haploid and only have one copy of each chromosome (in humans, 23 total).
  • A pair or set of identical chromosomes are called homologous chromosomes.
  • The full set of chromosomes (in humans, all 23) is referred to as the karyotype.

Whilst the number of genes = the complexity of species, the same rule is not true for the number of choromosomes as any particular chromosome may have a greater amount of DNA in it, and so any number of genes on it.

  • Also worth noting is that DNA contains sections of junk DNA which has a purely structural role – in humans only 9-27% is coding DNA (so only 9-27% can be used to synthesise proteins, the rest of the DNA strand is there to make sure the DNA can be stored correctly in chromosomes etc).

Fig 1 - high resolution image of a Chromosome showing different areas.

A Chromosome is made up of 2 chromatids joined at the centromere (also the point where spindle fibres attach during cell replication & division). There are 46 chromosomes (23 pairs) in a human; as shown.

Fig 2 - Showing Karyotype of Human Male

Note how there is no chromosome pair 23 on Fig 2. This is because the X and Y (or the sex chromosomes) form the final pair. Having 1 X and 1 Y chromosome results in a Male while 2 X chromosomes result in a female.

Offsite reading:

  1. http://www.ebi.ac.uk/2can/bioinformatics/dna.html – More detailed history of DNA discovery from the European Bioinformatics Institute.