Preparation for the “WDYTYA Practical”.

DNA Extraction

To work on DNA in lab – must extract it from cell. Do this using protocols tailored to the type of tissue the DNA is stored
in – extracting DNA from bacterial cell starts differently than mammalian cells or plant cells. This also differs depending
on the purity of DNA required – whether contaminants are a problem or not – the purer it is – the longer it will take.

Most protocols have these principles in common: cell lysis + protein removal, precipitation, washing of DNA and
resuspension.

Cell Lysis and Protein Removal

Must break open cells to get to DNA – involves getting through cell wall/membrane and organelle membrane (if present
in cell). As DNA is packaged in proteins – need to remove these. May want to remove contaminating cellular proteins
from prep.

Typically start with lysis buffer – within these there are detergents such as SDS (this along with heat help break up fats,
opening up lipid membrane/cell). Buffer also has salts – help to remove proteins from membrane by altering their
polarity – makes them clump together – easier to separate them from DNA. May also find EDTA - this reagent is involved
in protecting DNA from DNases by stopping them working. Does this by causing chelation (“grabbing”) of divalent ions
such as Mg2+ (ion with a valency of 2) – significant as DNases use magnesium ions as cofactors in their functioning –
without these – wont work. Finally, might find “tris” – this is a buffer to stabilise alkaline pH.

Proteins are denatured and dissolved from the detergents and salts – other ingredients may assist in protein removal.
Depends on if you want to dissolve them into a solution or simply denature. Proteinase K – degrade proteins. Chaotropic
salts (e.g. guanidine hydrochloride) – weaken the forces holding the protein together (such as Van Der Wall forces and
Hydrogen bonds). Ammonium acetate – reagent that causes proteins to precipitate – come out of solution – taking solid
form rather than aq-dissolved form – solution needs to be centrifuged and unwanted proteins will form a pellet

Precipitation

Following removal of DNA and protein from cell – must precipitate out DNA from solution just as ammonium acetate did
to proteins – done by added Isopropanol or ethanol – makes nucleic acids turn into a solid form. DNA is then separated by
centrifugation (if in microcentrifuge tube), or by using a silica matrix if using the column method (discussed further in
term). DNA is insoluble in alcohol – isopropanol and ethanol leads to aggregation of DNA molecules out of solution. This
means in centrifugation – DNA will then form the pellet. If using silica matrix – DNA binds to silica matrix

Wash

Once DNA/proteins separated in solution, the pellet or matrix needs to be washed to remove contaminants. This is
typically done in 70% ethanol solution. Use 70% rather than the 100% in previous step as the salts you are trying to
remove wont dissolve in 100% ethanol. If this isn’t done – leads to disruption later in process. Wash solution will either
pass through the matrix column or simply wash the pellet by re-suspending and centrifuging again.

Resuspension

To resuspend DNA back into solution (easier to work with) – simply add water – DNA is soluble in water. Must be purified
water and DNase free to protect the DNA from degradation. Other popular solution – TE buffer – includes Tris and EDTA –
serve same purposes as before to protect DNA.
Types of Nucleotide Triphosphates

Fig 1 – nucleotides before incorporated into DNA – nucleotide triphosphates. Ribonucleoside Triphosphates – building
blocks to make RNA. Deoxyribonucleoside triphosphate (dNTP) – DNA building block. Dideoxyribonucleoside
triphosphate. Difference are in hydroxyl group at 3’ end. NTP – has 2 OH groups (2’ and 3’ carbons). dNTP – has 1 – 3’.
ddNTP has none – has no potential to add any additional nucleotides onto the chain – need the OH groups.

Nucleoside triphosphates specified based on the base they have – dATP, dTTP etc. When ddNTP is used in DNA chain –
it will terminate as there is no OH group on the sugar.

Formation of phosphodiester bond

Bond found between oxygen atom in OH and phosphorous atom in another nucleotide’s phosphate group. This occurs
in the 5-3’ direction as OH is on 3’ end of nucleotide. Formation of this phosphodiester bond causes the loss of 2
phosphate groups. However, if ddNTP added – lack of OH group – no oxygen to form bond with phosphorous – chain
stops extending.

Sanger Sequencing of DNA

Uses chain termination principles (ddNTPs) – to our advantage. Start with reaction similar PCR – instead of adding
dNTPs – instead add mixture of dNTPs and ddNTPs so they chain will stop in certain places. Then undergo thermal
cycling similar to PCR. Chain termination sequencing only works with one primer at a time, rather than the 2 in PCR –
this is focusing on extension of one strand at a time. This is so we can work out the sequence of this particular
template strand.

To do this – primer could be radioactively labelled with radioactive phosphorous. In order to keep track of the product
– this is helpful later on. Cycle sequencing reaction started – with radioactive primer, DNA polymerase and a mixture
of dNTPs and a small amount of a particular type of ddNTP (i.e., it may be ddATP or ddCTP – must be just one, not the
others). This means whenever this ddNTP is added – the chain stops extending. However, because we have dNTPs as
well, when this ddNTP is added will differ in each cycle. With multiple chains being synthesised at a time – going to
produce chains (aka fragments) at different lengths – eventually you will chains that stop at every point your
nucleotide of interest is present in that template (for instance, every T) (fig 2)

In the old method – you would separate your template in 4 different reactions – each one adding normal
requirements for PCR alongside one of the 4 ddNTPs – in each of these reactions the process described above would
occur. All of these fragments are then ran on a gel electrophoresis – this allows us to read up the gel (starting with the
smallest as the chain is synthesised from 5’-3’) – as each band will only be separated by a single base (fig 3) – used to
find out the sequence of the entire template strand.

Note that the old method uses radioactively label primer and 4 different reactions. Today this is more sophisticated –
instead label the ddNTPs different colours with fluorescent label. This way 4 separate reactions aren’t needed. Add
regular dNTPs alongside all 4 ddNTPs at low concentration – cycle sequencing reaction occurs. Use gel electrophoresis
to separate the different size strands. Here we use a capillary gel, as the fragments move down this gel – passes a
detector – shines laser onto ddNTPs exciting the fluorescent label – the colour that shines as a result tells you the
ddNTP present and therefore the sequence of DNA. This is presented on a chromatograph – fig 4 – shows how much
fluorescence is present at a particular strand length.

The human genome project cost $3 billion dollars at took 13 years to complete (1990-2003) – used old method to read
entire human genome. Now can sequence a complete human genome within 24 hours – new methods that go beyond
sanger sequencing (Sanger sequence done couple hundred bp at a time) – next generation sequencing. Costs around
£5000 to get genome sequenced.
Human Evolution and Migration

Fig 5 – migration of ancient Humans. We (Homo sapiens) – originated in East Africa 200,000 years ago. 100,000 years
ago – spread to Middle East and Asia, 40,000 years ago into Europe. Garlands settled most recently. Over these periods
of time – DNA acquires mutations. DNA of individuals who originated in Africa genome therefore differs from those in
South America. Long and slow process but enough mutations acquired to allow identification of different populations
of humans via the mutations in their DNA

Nuclear DNA

You can get different information about heritage from nuclear and mitochondrial (mt) DNA – down to their
characteristics and how they are inherited. Nuclear DNA – in nucleus – 23 chromosomes – half from father, half from
mother. In turn – they got their nuclear DNA from their grandparents. Going back 5 generations – 1/32 of your nuclear
genome contributed – probably less due to recombination. Therefore we share little nuclear DNA with relatives – very
difficult to keep track of over long time periods.

MtDNA

Easier to track the inheritance of this DNA – no recombination due to prokaryotic origin – passed on as is. Maternally
inherited – also easier to track to past.

mtDNA – circular and much smaller than human genome – 16,569 bp compared to 3 billion in DNA. There is a 900bp
region where the sequence is more varied among individuals – called the D-loop or hypervariable region (fig 6). mtDNA
codes mostly for functional products – rRNAs and tRNAs for protein synthesis and proteins for the respiratory chain.
Mutations acquired in these regions – most likely fatal – evolution very quickly selects against these. D-loop region – no
functional genes – mutations are therefore not selected against – give good record of what happens over time.
Sequence of mtDNA starts right in middle of D-loop – splits the D-loop into 2 HVRs.

Advantages of mtDNA – can be amplified from old/degraded samples such as fossils. Maternal relatives will have the
same sequence. Disadvantages of mtDNA – not specific to an individual. Random individuals may have same sequence
– cant be used for forensic identification.

Using mtDNA to track migration

Fig 7. Sequencing mtDNA of people across globe – show which mutations occur where – this is used to create
phylogenies. Work backwards – which sequence is the simplest – from this which sequences have deletion/addition
mutations – must be descendants.

Haplogroups – a collection of individuals with the same mutations within their mtDNA. Haplogroup “L” – last common
matrilineal ancestor haplogroup – mutations accumulate from this. Following where these mutations are found – this
gives an idea of how we migrated.
Genetic Diversity

The total number of genetic differences within a population or species. 3 vital reasons why it is important: evolution
cannot happen without genetic diversity – something must be selected for or against; needed to protect endangered
species – more likely to inbreed – lowering diversity and therefore making them vulnerable to selection pressures; also
needed to improve food/crops we eat – need diversity in germplasm to create new combinations via breeding.

Bottlenecks reduce genetic diversity drastically. Only a small numbers of a particular species survive event or become
isolated – overtime population size will increase – only limited by the environment, however genetic diversity will not as
this requires accumulations of mutations over generations.