F215 Biology: Genomes and Genomics
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A genome is all of the genetic information within an organism. It is made of DNA divided into a
definitive number of chromosomes.
Genomics is the study of the whole set of genetic information in the form of the DNA base
sequences that occur in the cells of a particular species.
The human genome contains 3 billion bases. 22,000 genes are present; only 1.5% of bases are
coding bases (that it, bases which code for a particular protein). The remaining DNA(often
mistakenly referred to as ‘junk DNA’) is still important: control sequences, ‘fossil genes’ from our
evolutionary past, parasitic DNA that can jump around the genome, inactive viral genomes and
simple repetitive sequences (eg. TATATA) that are often found at the end of chromosomes
(‘telomeres’).
Exons are coding regions of chromosome; introns are non-coding regions.
Comparative genomics: when we know the sequence of bases in a gene of one organism we can
compare genes for the same proteins across a range of organisms. This comparative gene
mapping has 5 main applications:
o Identification of genes for proteins found in all or many living organisms gives clues to
the relative importance of such genes to life.
o Comparing genes of different species shows evolutionary relationships. The more DNA
sequences the organisms share, the more closely related they are likely to be. When
comparing closely related species, we need to look at fast evolving genes that are
unique to the chosen species – like genes coding for their cells’ antigens. Species that
are more distantly related must be compared by looking at genes that code for proteins
fundamental to life processes that rarely change, that all organisms have in their
genome, like genes for ribosomes and tRNA.
o Comparing genomes can reveal the effects of mutations to DNA. Mutations in yeast
cells have been found that cause unusually fast mitosis. The same gene has been found
in humans, so experiments on yeast can be used to find drugs that could be useful to
humans.
o Comparing genomes of pathogenic and similar non-pathogenic organisms can be used to
identify the base sequences that are most important in causing disease. This can lead
to identifying targets for more effective drugs and vaccines.
o Analysing DNA of individuals can reveal mutant alleles or the presence of
allelesassociated with increased risk of particular diseases like heart disease or cancer.
Genetic fingerprintingis a technique that can be used to identify individuals. It works because
every individual has a unique sequence of nucleotide bases in their genomes.
In order to carry out genetic fingerprinting, DNA samples are cut at specific sequences of
nucleotides. DNA samples from different individuals will be cut into a different number and
different size of ‘restriction fragments’, so when the DNA samples are separated according to
size, a person can be identified from the unique fragment pattern.
The three key ingredients for genetic fingerprinting are: restriction enzymes, gel electrophoresis
and gene probes.
Restriction enzymes (restriction endonuclease enzymes) cut DNA at a specific base sequence
known as a restriction site. Restriction enzymes are produced naturally by prokaryotes where it
evolved to provide a defence mechanism against invading DNA viruses called
F215 Biology: Genomes and Genomics
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bacteriophages.The bacteria’s own DNA is methylated to protect it from the effects of the
enzyme. Restriction enzymes recognise a specific sequence of nucleotides and produce a double
stranded cut in the DNA. Two incisions are made, one through each sugar-phosphate backbone
of the DNA double helix. Restriction enzymes usually make a staggered cut through the DNA,
leaving sticky ends.
Any two people will have small differences in their DNA. Each person’s DNA will be cut into a
different number and size of fragments; these are ‘Restriction Fragment Length
Polymorphisms’. The mix of fragments will be the person’s fingerprint.
Gel electrophoresis separates DNA fragments based on their size. DNA fragments are pipetted
into a well at one end of an agarose gel with a buffer solution to stabilise pH and ensure that
DNA stays double stranded. A size standard is placed into the well at the end of the gel to allow
for the quick estimation of the size of unknown fragments in other lanes. DNA fragments
migrate to the positively charged anode (DNA is negatively charged due to the phosphate
groups). Small fragments migrate quicker and further than larger fragments.
The Southern Blot technique is used to create an exact but more durable copy of the gel. A
nylon sheet is placed over the DNA fragments. Paper towels are placed over the top so DNA
fragments are drawn out of the gel and onto the nylon sheet.
DNA probes are a short single stranded piece of DNA (50-80 nucleotides) complementary to a
piece of DNA being investigated. The probe is labelled in one of two ways: using a radioactive
marker that can be revealed by exposure to photographic film or by using a fluorescent marker
that emits a colour on exposure to UV light. DNA fragments are made single stranded by adding
alkali. When added to DNA fragments, the probe will anneal (form hydrogen bonds) with
complementary sequences and we are then able to identify the position of specific genes on the
gel.
Genetic fingerprinting is used in paternity testing to identify a child’s biological father and in
crime scene investigations to find if a suspect was present at the scene of a crime.
DNA Chips and Microarrays: this technology allows us to rapidly determine which genes are
expressed by a cell or tissue. When a gene is expressed, it is transcribed into mRNA. mRNA from
the tissues in question is isolated and then converted into complementary strands of DNA
(cDNA) by the enzyme reverse transcriptase. To distinguish between the cDNA of the different
cells, they are fluorescently labelled. A DNA chip contains 60,000 or more different DNA
sequences called probes. The probes are single stranded represent a unique region of a gene in
the genome. cDNA samples are mixed together and added to the chip. cDNAs that are
complementary to probes on the chip will hybridise with DNA and stick to that location on the
chip. Unbound cDNAs are washed away. A scanner detects patterns of hybridisation by sensing
the fluorescent signals. Each area of the chip contains a known DNA sequence, so the identities
of the hybridising cDNA can be determined. Using this, we can find which genes are expressed
differently in cancerous or diseased tissue, so we may be able to design better treatment
strategies.
Polymerase chain reaction: this reaction is able to make thousands of copies of just one
particular gene from small quantities of mixed DNA. If scientists have a limited amount of DNA
to work with, they need to replicate it so they have 1000s of copies before the investigation
begins. PCR is useful when we need to amplify DNA from a crime scene, or when we need to
replicate DNA before genetic engineering or before gene therapy.
PCR needs 3 key ingredients:free DNA nucleotides, primers and Taq polymerase enzyme.
F215 Biology: Genomes and Genomics
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Free nucleotides must be activated with 3 phosphate groups.
Taq polymerase is the DNA polymerase enzyme from the thermophillic bacteria Thermus
Aquaticus. This bacterium can survive at temperatures of 50-80° degrees. It is thermostable – it
allows DNA to be heated to melting point and the enzyme is not denatured.
Primers are short artificial nucleotide sequences, 4-20 bases in length, which are
complementary to the ends of the gene of interest. Primers identify the start point for DNA
replication by Taq polymerase. Taq polymerase would be unable to bind directly to single
stranded fragments.
The procedure in Polymerase Chain Reaction:
1. Heat DNA to 95° to break the hydrogen bonds holding the DNA double helix together,
so making the samples single stranded.
2. Add primers and reduce temperature to 55° allowing the primers to anneal to the ends
of the gene of interest.
3. Heat to 72° to allow the Taq polymerase to bind. The DNA polymerase enzyme adds
free nucleotides to the unwound DNA, extending the double stranded section.
4. Repeat the process; the amount of DNA increases exponentially.
DNA sequencing – based upon interrupted Polymerase Chain Reaction and Electrophoresis
DNA sequencing uses the principles of PCR and electrophoresis. The DNA strand to be
sequenced is copied millions of times using Taq polymerase, primers and free nucleotides (just
as in PCR). The DNA fragments produced are then sorted according to size by electrophoresis.
Within the sequencing mixture, some of the nucleotides are fluorescent dideoxynucleotides.
These nucleotides are modified and if they are added to the growing chain, the DNA polymerase
stops the replication reaction and the DNA strand will end this fluorescently-marked
dideoxynucleotide.
The DNA strand is heated to 95° to denature the DNA making it single stranded.
Mixture is cooled to 55° so that primers can anneal at the 3’ end of the template strand.
Heat the mixture to 72° so that Taq polymerase will attach. The polymerase enzyme will add
nucleotides according to complementary base pairing.
If a fluorescent dideoxynucleotide is incorporated into the growing chain, the replication
reaction will stop. The fragment will remain this length with the fluorescent dideoxynucleotide
as the final base.
As the reaction proceeds, many molecules of DNA are made. The
fragments generated vary in size. In each case, the final added
nucleotide is tagged with a specific colour.
An automated DNA sequencer separates the fragments by size. This is
like a vertical gel electrophoresis apparatus with a laser at one end. The
laser will read the colour sequence of the dideoxynucleotides at the end
of each fragment.
We can now identify the base type at the end of each fragment and the
base’s position along the DNA molecule.
F215 Biology: Genomes and Genomics
Sequencing a genome – BACs
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Genomes are first mapped to identify which chromosome or which section of the chromosome
they have come from. The location of microsatellites are used – these are short runs of
repetitive sequences of 3-4 base pairs found in several thousand locations in the genome.
Samples of the genome are sheared (mechanically broken) into smaller section of around 10000
base pairs.
These sections are placed into bacterial artificial chromosomes (BACs) and transferred into the
cells of E.Coli bacteria. BACs are man-made pieces of DNA that can replicate inside a bacterial
cell.
As the bacterial cells grow in culture, many clones of the DNA sections are produced. These cells
are referred to as clone libraries.
In order to sequence a BAC section, cells containing specific BACs are taken and cultured. The
DNA is extracted and restriction enzymes are used to cut it into a number of smaller fragments.
Different restriction enzymes are used on a number of different samples to give different
fragment types.
The fragments are separated by electrophoresis. Each fragment is sequenced using an
automated process. Computer programmes then compare overlapping regions from the cuts
made by different restriction enzymes in order to reassemble the whole BAC segment sequence.