DNA and the Genome Key Area 8a Genomic Sequencing

(a) Genomic sequencing Learning Intentions By the end of this topic you should be able to: (a) Genomic sequencing Define ‘genomic sequencing’ Name 2 methods of genomic sequencing Describe the outcome of comparing the genome from different species Explain what ‘many genes are conserved across different organisms’ means

Genomic Sequencing

Genomics Genomics is the study of genomes and involves determining the sequence of nucleotide bases along an organisms DNA Genomic sequencing involves determining the sequence of nucleotide bases for individual genes and entire genomes Computer programs can be used to identify base sequences by looking for sequences similar to known genes. This allows scientists to relate gene sequences to function To compare sequence data, computer and statistical analyses (bioinformatics) are required

Genomic Sequencing The sequence of nucleotide bases provides a code which is unique to an organism Remember, the DNA sequences code for an amino acid sequence which then codes for proteins

Genomic Sequencing DNA sequencing allows the order of bases on an unknown sequence of DNA to be determined. The first stage is to amplify the DNA fragment whose sequence is to be determined using PCR As previously discussed, PCR requires the following: DNA template Primers: small (20bp) sequence of DNA complementary to one of the DNA strands of template DNA nucleotides (A T C G) Taq Enzyme (polymerase) Buffer Thermal cycler machine

Genomic Sequencing However, into the mix a special modified set of nucleotides called dideoxynucleotides with fluorescent dyes attached are also required These modified nucleotides are designed so they prevent further extension of DNA chains during PCR The four dideoxynucleotides are: ddA, ddT, ddC, ddG

Dideoxynucleotides (ddNTP) These modified nucleotides lack the 3’ Hydroxyl (OH) group which prevents more phosphodiester bonds forming Normal DNA nucleotide with Adenine base (dA) Dideoxynucleotide with Adenine base (ddA)

This modified ddNTP lacks the 3’ OH and so cannot form a bond with the next nucleotide

Genomic Sequencing There are 2 possible methods of sequencing DNA: Sanger Method Dye-terminating Sequencing

Sanger sequencing Primer radioactively labelled Four experiments set up Each tube contains ALL normal dNTPs (+ DNA polymerase) ONE TYPE of ddNTP is placed in each tube

Sanger sequencing Different length fragments produced in each incubation Gel electrophoresis each ddNTP in a different lane and then blot and expose to photographic film

Sanger sequencing Can read the sequence from the blot (bottom to top as smallest fragments run furthest) Remember to convert to complimentary sequence!

Dye-Terminating Sequencing The PCR is set up and runs as normal except there is only 1 mix and it contains the 4 types of ddNTP (each with a different coloured fluorescent dye) As the unknown sequence is being replicated, it is random whether a normal nucleotide or a modified dideoxynucleotide is added to the growing chain If a normal nucleotide is added, the replication of the DNA moves onto the next base If a dideoxynucleotide is added, the chain cannot get bigger By the end of the PCR there should be a mix of different sized fragments of every possible length The fragments are then separated by gel electrophoresis

Tag each ddNTP with a different colour fluorescence dye so each fragment produced has a different coloured tag at its chain terminating ddNTP Separate fragments by electrophoresis (run in one lane) Optical reader on sequencer identifies fluorescent tag on final bases

Dye-terminating Sequencing The electrophoresis requires a special machine called a sequencer As each fragment runs down through the gel it passes an optical reader which identifies the fluorescent dye on the ddNTP at the end of each fragment Special software then convert this information into the DNA sequence

A laser is fired as each fragment passes it The laser excites the dye on the end of each ddNTP causing it to fluoresce The detector picks up the order of the different dyes as they pass which allows the DNA sequence to be determined

Remember: The smaller fragments run further so you start reading the DNA sequence from the bottom of the gel You still have to convert the sequence back to its complementary strand to obtain original DNA sequence However, automated software do all this for you nowadays

This is what the computer software will produce after the unknown DNA sequence has been run through the sequencing machine

Genomic Sequencing The sequencing of an organisms DNA requires specific chemicals and technology A special type of enzyme called a restriction endonuclease is required Each endonuclease recognises a specific short sequence of bases on DNA called a restriction site and each time it encounters that restriction site it ‘cuts’ the DNA

The two restriction endonucleases above each recognise a different short specific DNA sequence and cut that sequence as shown

Genome Shotgun Approach When investigating an organisms genome it is first cut into sections using endonucleases 2 copies of the same genome are cut with different endonucleases. As they recognise and cut different sequences of DNA, the fragments produced from each endonuclease will have regions that overlap at their ends These fragments are put into 2 libraries and can be used to sequence the organisms DNA

Multiple copies of the genome made by PCR 2 (or more) endonuclease are used on separate copies of the genome creating multiple fragments Each fragment is sequenced and computer programs used to match overlapping fragments so sequence of genome is obtained

The original DNA is cut using different endonuclease enzymes and put into different ‘libraries’ A fragment from the first library is sequenced and the end of the sequence used to produce a probe (complimentary) The probe is then labelled so it can be identified and put into the second library As the probe sequence will be complimentary to the overlap region of one fragment in the second library, this fragment can be identified, sequenced and a new probe made which is put back into the first library and the whole procedure begins again

Comparative Genomics

Human Genome Project Scientists from different countries have worked together since the early 90s in a combined effort to sequence the whole of the human genome With over 3 billion nucleotides this was a massive undertaking but as the project proceeded, the massive leaps forward made with DNA equipment and analysis programmes made this a lot easier and faster than was first thought. The project used the shotgun approach to sequence DNA from a small number of human donors At the same time, DNA from other species were also being sequenced

Genomes from Different Species Other species genomes that were sequenced include: Viruses and Bacteria – most of these were disease causing and so were sequenced to look for cures or treatments Pest species – species such as mosquitos which act as a vector for malaria and which destroy crops Model organisms – important for research as they posses genes equivalent to human genes and so experiments on them may lead to an understanding of faulty genes in humans and possible provide data for new treatments

Comparing Genomes Comparison of genomes reveals that many genes are highly conserved across different organisms. Comparative genomics uses the sequenced genomes of different species to compare: Members of the same species Members of different species Cancerous v Normal cells

Members of the Same Species This allows the likes of a harmless strain of bacteria to be compared to a disease causing strain Any differences between the two genomes is the cause of the illness

Single Nucleotide Polymorphisms (SNPs) When the DNA sequence varies by one base pair between genomes it is called a single nucleotide polymorphism (SNP) SNPs usual arise as a result of a point mutation where one base is substituted for another Over 1 million SNPs have been identified and located in the human genome and it is hoped this SNP map will help identify genes and mutations associated with disease

individual individual individual individual When the same chromosome from different individuals is sequenced and bioinformatics used to compare them, different SNPs can be identified The above example shows 3 different SNPs when comparing the same chromosome from 4 different individuals

Members of Different Species Comparison of genomes across different species also helps identify highly conserved genetic sequences – the same or very similar DNA sequence is found in the different species These sequences tend to code for the active sites of essential enzymes and so have been positively selected for over time The comparison of genomes and these conserved sequences help determine how close or distant the relationship between two species are The greater the number of conserved DNA sequences there are between two species, the more closely related the two species are

Cancerous v Normal Cells If cancerous and normal cells are obtained from the same individual they can be used to identify the mutations which cause a healthy cell to divide uncontrollably and form a tumour