What into the techniques of genomics such as DNA

What is Genomics

Genomics is a study
of the genomes of organisms. It main task is to determine the entire sequence
of DNA or the composition of the atoms that make up the DNA.

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Introduction

The research of
genomics initially begun when Frederic Sanger (a British biochemist) sequenced
the complete genomes of a virus and the mitochondrion within. Other scientists
were inspired to continue research into the techniques of genomics such as DNA
sequencing and gene mapping, eventually creating the Human Genome Project. The
knowledge of the human genome sequence has been used to further investigate genomics,
specifically, to describe gene functions and interactions during various
conditions such as cancer.

The application of
genomics within cancer, works, by evaluating the genes in a target location of
cancerous tissue. In this way, mutated genes can be identified and compared to
those of which have been inherited.

 

 

Techniques

Cancer genomes
frequently alter their chromosomal structure by amplification, deletion,
translocation and/or inversion. Many of these alternations change the amino
acid chain within the genome, thus altering the genes in many ways that may be
critical to cancer onset or progression. Therefore, the analysis of structural
variation (SV) in tumour genomes have been recently performed. Initially, these
studies were conducted using single nucleotide polymorphism(SNP) array data
sets, where tumour and normal genomic DNA were compared, and any large-scale
amplification or deletion signals were detected. The use of this technique has
led to the discovery of new oncogenes in ovarian cancer, melanoma and lung
carcinoma.

 

However, according to a sequencing
specialist at Washington University, sequencing can map the locations of
insertions and duplications with more precision and can catch deletions that
might have gone undetected by an array. Scientists at Washington university
used sequencing to detect overlapping deletions in a breast cancer that had
spread to other parts of the body. The deletions spanned the region containing CTNNA1, a gene thought to
suppress the spread, of cancer.

 

 

Genomic techniques, such as PCR and Sanger sequencing (SGS) to
discover mutations in tumour genomes was initially proven to be a powerful
approach. Studies using this method have successfully identified key somatic
mutations in cancer genomes.
An article published by the journal, Nature, demonstrates how targeted gene
re-sequencing can contribute significantly to our understanding of the types of
genes carrying mutations in each cancer type.

SGS works by adding a modified nucleotide, dideoxynucleotide(ddNTP),
to the DNA chain which lacks a 3′ OH. Thus, when attached, the ddNTP terminates
DNA strand elongation. Each ddNTP may be fluorescently labelled for detection
in sequencing machines.  

 

 

 

 

 

This traditional Sanger sequencing was found to be too labour
intensive to then perform on the entire human genome. It is not very efficient
for the following reasons:

·        
Had
to set up 4 mini reactions (one for each ddNTP)

·        
Had
to read the sequence by eye

·        
The
human genome has 3×10^9 base pairs whilst a ‘good’ run using Sanger gives you
~300.

 

Electrophoresis Capillary

A better method is to use capillary electrophoresis and
fluorescent labelling where ddNTP is labelled with a fluorescent base. No
longer need 4 separate reactions. Can run sample down one electrophoresis
capillary. A laser bean near bottom of column energises the fluorescence attached
to base. Flourescence detector identifies ddNTP. Sequence is “read” by a
computer.

Many
of the molecular diagnostic techniques within cancer require a seperation step
before detection, thus capillary electrophoresis can perform rapid and
efficient seperation within samples.

 

 

Illumina sequencing

·        
Within
this technique, sequencing DNA fragments are annealed to a slide.

·        
PCR
is carried out to amplify each read

·        
The
reads are separated into single strands to be sequenced.

·        
The
slide is flooded with nucleotides and DNA polymerase. These nucleotides are fluorescently
labelled, with the colour corresponding to the base. They also have a
terminator, so that only one base is added at a time.

Therefore, when attached, each nucleotide will light up a
different colour. To prevent a 2nd nucleotide attaching to the green
T below, the fluorescent dye produces a chain termination. Only until the
fluorescent probe has been cleaved by an enzyme will it be possible to add
another base.

Advantages of this method in cancer

·        
It is
very quick to identify the sequence of DNA fragments

·        
Large
read lengths of genomes

 

 

 

 

 

 

 

 

 

 

The advancements in next generation sequencing (NGS) methods
have improved the research into cancer diagnostics and treatment. Specifically, in breast cancer,
sequencing of cancer genomes has revealed new cancer-related genes which are
grouped into cancer-related pathways. For example, mutations of a gene in
breast cancer within the luminal A subtype have been found. This allows us to know
the general area of mutated gene, resulting in a faster and more efficient
treatment.

Unlike Sanger sequencing, PCR, and microarrays, using NGS
excludes certain limitations which affect the efficiency of the sequencing. For
example, microarrays can detect single nucleotide variants (SNVs), however they
have trouble identifying larger DNA characteristics. In contrast, NGS can
provide us with a wider view of the DNA genome, genetic recombination, and
other mutations. Therefore, NGS platforms serve as a good diagnostic tool to
help clinicians identify specific characteristics in each patient.

NGS has been implemented in cancer diagnosis and prognosis. One
example is where whole genome sequencing identified an insertion within DNA
that created a mutated gene for a patient with acute myeloid leukaemia. The
findings from the sequencing altered the treatment plan for the patient. By
sequencing the tumour genome of a patient, clinicians can target
patient-specific probes that uses DNA in the patient’s blood serum to monitor
the progress of a patient’s treatment and detect for any signs of relapse. The
discovery of more biomarkers and the development of target-therapies will be
essential in helping a clinician choose the best personalized treatment for his
or her patients.

 

 

 

Conclusion

Not everyone agrees. Some researchers argue that the
costs of cancer-genome projects currently outweigh the benefits. Prices are
poised to drop dramatically in the next few years as a new generation of
sequencing machines comes online, says Ari Melnick.

 

The effect of cancer genomics is palpable in every corner
of cancer research. However, its presence in the clinic is still limited. This
is in part because the practice of medicine demands a balanced assessment of
relative risk and benefit to the patients, which requires a level of evidence
that goes beyond scientific discovery.