DNA Sequencing Omar Alomair Homod Alherbi Fisal Alkulify.

DNA SequencingDNA Sequencing

Omar AlomairOmar Alomair

Homod Alherbi Homod Alherbi

Fisal AlkulifyFisal Alkulify

IntroductionIntroduction::

The term The term DNA sequencingDNA sequencing encompasses biochemical methods for encompasses biochemical methods for determining the order of the determining the order of the nucleotide bases, adenine, guanine, nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA cytosine, and thymine, in a DNA oligonucleotide. oligonucleotide.


The sequence of DNA constitutes the The sequence of DNA constitutes the heritable genetic information in nuclei, heritable genetic information in nuclei, plasmids, mitochondria, and plasmids, mitochondria, and chloroplasts that forms the basis for chloroplasts that forms the basis for the developmental programs of all the developmental programs of all living organisms. living organisms.


Determining the DNA sequence is Determining the DNA sequence is therefore useful in basic research therefore useful in basic research studying fundamental biological studying fundamental biological processes, as well as in applied fields processes, as well as in applied fields such as diagnostic or forensic such as diagnostic or forensic research. research.


The advent of DNA sequencing has The advent of DNA sequencing has significantly accelerated biological significantly accelerated biological research and discovery. The rapid research and discovery. The rapid speed of sequencing attainable with speed of sequencing attainable with modern DNA sequencing technology modern DNA sequencing technology has been instrumental in the large-has been instrumental in the large-scale sequencing of the human scale sequencing of the human genome. genome.

How do we Sequence How do we Sequence DNADNA

First you need to know a First you need to know a few key termsfew key terms::PlasmidPlasmidA 'plasmid' is a small, A 'plasmid' is a small, circular piece of DNA that circular piece of DNA that is often found in bacteria. is often found in bacteria. This innocuous molecule This innocuous molecule might help the bacteria might help the bacteria survive in the presence of survive in the presence of an antibiotic, for an antibiotic, for example, due to the example, due to the genes it carriesgenes it carries . .

Key termsKey terms

GenomeGenome

In biology the In biology the genomegenome of an organism is its of an organism is its whole hereditary whole hereditary information and is information and is encoded in the DNA encoded in the DNA (or, for some viruses, (or, for some viruses, RNA). This includes RNA). This includes both the genes and the both the genes and the non-coding sequences non-coding sequences of the DNAof the DNA

Key termsKey terms

• PlasmidPlasmid • To scientists, however, plasmids are To scientists, however, plasmids are

important because (i) we can isolate important because (i) we can isolate them in large quantities, (ii) we can them in large quantities, (ii) we can cut and splice them, adding cut and splice them, adding whatever DNA we choose, (iii) we can whatever DNA we choose, (iii) we can put them back into bacteria, where put them back into bacteria, where they'll replicate along with the they'll replicate along with the bacteria's own DNAbacteria's own DNA

Key termsKey terms

BACBAC

The term 'BACThe term 'BAC" " is an acronym for is an acronym for 'Bacterial Artificial Chromosome', and 'Bacterial Artificial Chromosome', and in principle, it is used like a plasmidin principle, it is used like a plasmid. . We construct BACs that carry DNA We construct BACs that carry DNA from humans or mice or wherever, and from humans or mice or wherever, and we insert the BAC into a host we insert the BAC into a host bacteriumbacterium. .

Key termsKey terms

BACBAC

As with the plasmid, when we grow As with the plasmid, when we grow that bacterium, we replicate the BAC that bacterium, we replicate the BAC as wellas well. . Huge pieces of DNA can be Huge pieces of DNA can be easily replicated using BACs Using easily replicated using BACs Using BACs.BACs.

Key termsKey terms

PrimerPrimer

A primer is a strand of nucleic acid that A primer is a strand of nucleic acid that serves as a starting point for DNA serves as a starting point for DNA replication. replication.

Key termsKey terms

VectorVector

The 'vector' is The 'vector' is generally the basic generally the basic type of DNA type of DNA molecule used to molecule used to replicate your DNA, replicate your DNA, like a plasmid or a like a plasmid or a BAC .BAC .

Key termsKey terms

InsertInsert

The 'insert' is a The 'insert' is a piece of DNA we piece of DNA we have purposely put have purposely put into another into another ((a a 'vector''vector') ) so that we so that we can replicate itcan replicate it. .

DNA SequencingDNA Sequencing

DNA PREPARATIONDNA PREPARATION► DNA needs to be purified DNA needs to be purified

from cells. First, the cells from cells. First, the cells and their nuclei are broken and their nuclei are broken open. This can be open. This can be accomplished by accomplished by mechanical methods, such mechanical methods, such as grinding, or by chemical as grinding, or by chemical methods that break apart methods that break apart cell membranes. The DNA cell membranes. The DNA floating around in this floating around in this “soup” is still coated with “soup” is still coated with protective proteins. The protective proteins. The DNA can be selectively DNA can be selectively removed from this soup by removed from this soup by precipitating it and DNA-precipitating it and DNA-binding proteins can be binding proteins can be cleaned away.cleaned away.

Automated DNA Automated DNA SequencingSequencing

DNA PREPARATIONDNA PREPARATION► Very large pieces of DNA, Very large pieces of DNA,

such as whole such as whole chromosomes or chromosomes or genomes, are cut into genomes, are cut into smaller pieces and stored smaller pieces and stored in vectors in vectors ((plasmidsplasmids)), , which are larger pieces of which are larger pieces of DNA with the ability to be DNA with the ability to be reproduced when placed reproduced when placed in host cells such as in host cells such as bacteriabacteria..

Automated DNA Automated DNA SequencingSequencingDNA PREPARATIONDNA PREPARATION► Bacteria containing a vector Bacteria containing a vector

are placed in culture are placed in culture medium, where they medium, where they multiply a million-fold or multiply a million-fold or more. Each time a more. Each time a bacterium divides, the DNA bacterium divides, the DNA vector placed inside is also vector placed inside is also copied. In this way, the copied. In this way, the target DNA can be target DNA can be multiplied exponentially. multiplied exponentially. Each of the copied DNA Each of the copied DNA pieces is called a clonepieces is called a clone..


SEQUENCING REACTIONSEQUENCING REACTION► The sequencing reaction itself consists of four steps, The sequencing reaction itself consists of four steps,

which will be covered in detail in this section. First, the which will be covered in detail in this section. First, the double-stranded DNA is separated into single strands, double-stranded DNA is separated into single strands, and a small starter piece of DNA called a primer binds and a small starter piece of DNA called a primer binds to one of the strands, called the template strandto one of the strands, called the template strand..


SEQUENCING REACTIONSEQUENCING REACTION► In the extension step, a new DNA strand is In the extension step, a new DNA strand is

made that is complementary to the made that is complementary to the template strand. Starting at the primer, DNA template strand. Starting at the primer, DNA polymerase uses the template strand as a polymerase uses the template strand as a guide to recreate the second DNA strand.guide to recreate the second DNA strand.


SEQUENCING REACTIONSEQUENCING REACTION► The termination step is the key to the The termination step is the key to the

sequencing reaction. Strand extension is halted sequencing reaction. Strand extension is halted by the incorporation of a dye-labeled by the incorporation of a dye-labeled terminator nucleotide, which identifies the terminator nucleotide, which identifies the base at the position where strand extension base at the position where strand extension stopped. When many strand termination stopped. When many strand termination reactions are performed together, each of the reactions are performed together, each of the bases in a DNA strand can be identified.bases in a DNA strand can be identified.


STRAND SEPARATIONSTRAND SEPARATION DoubleDouble--stranded DNA needs to be denatured, stranded DNA needs to be denatured,

or separated into single strands, before it can or separated into single strands, before it can be sequencedbe sequenced. . This process is accomplished This process is accomplished by heating the DNA, which disrupts the by heating the DNA, which disrupts the hydrogen bonds and VanDerWaals forces that hydrogen bonds and VanDerWaals forces that hold the two chains of DNA together in a hold the two chains of DNA together in a double helixdouble helix. .


PRIMER ANNEALINGPRIMER ANNEALING► A small single-stranded DNA piece of about 20 A small single-stranded DNA piece of about 20

bases, called an oligonucleotide, is annealed to the bases, called an oligonucleotide, is annealed to the denatured template strand. This oligonucleotide is denatured template strand. This oligonucleotide is needed to “prime” the next step, DNA extension. needed to “prime” the next step, DNA extension.

Automated DNA Automated DNA SequencingSequencingPRIMER ANNEALINGPRIMER ANNEALING► the two DNA strands that were separated in the the two DNA strands that were separated in the

preceding step could just snap back together. This preceding step could just snap back together. This is avoided by using a rapid cooling process, which is avoided by using a rapid cooling process, which gives the small nucleotides an advantage over long gives the small nucleotides an advantage over long DNA strands in annealing. In addition, a large DNA strands in annealing. In addition, a large excess of primers is used to again ensure that the excess of primers is used to again ensure that the primers will out-compete the complementary DNA primers will out-compete the complementary DNA strand for annealing to the template.strand for annealing to the template.

Automated DNA Automated DNA SequencingSequencingPRIMER ANNEALINGPRIMER ANNEALING► The oligonucleotide primer must be of The oligonucleotide primer must be of

complementary sequence to the template complementary sequence to the template strand in order to bind by base-pair interactions.strand in order to bind by base-pair interactions.


PRIMER EXTENSIONPRIMER EXTENSION

► During the extension During the extension phase, a bacterial DNA phase, a bacterial DNA polymerase enzyme polymerase enzyme begins assembling a begins assembling a new DNA chain from new DNA chain from the individual the individual nucleotide building nucleotide building blocks, or dNTPs, blocks, or dNTPs, provided in the provided in the reaction mixture.reaction mixture.

Automated DNA Automated DNA SequencingSequencingPRIMER EXTENSIONPRIMER EXTENSION► The nucleotides are added in the order specified The nucleotides are added in the order specified

by the complementary bases in the template by the complementary bases in the template strand. DNA polymerase cannot start copying a strand. DNA polymerase cannot start copying a template strand without a small piece of DNA to template strand without a small piece of DNA to start the extension process. This is why the start the extension process. This is why the primer was added in the previous step.primer was added in the previous step.


CHAIN TERMINATIONCHAIN TERMINATION► The reaction mixture also contains small The reaction mixture also contains small

amounts of each of the 4 dideoxynucleotides, amounts of each of the 4 dideoxynucleotides, or “ddNTPs,” which lack the 3'-hydroxyl group or “ddNTPs,” which lack the 3'-hydroxyl group necessary for chain extension. Whenever a necessary for chain extension. Whenever a dideoxynucleotide is incorporated into a dideoxynucleotide is incorporated into a growing DNA chain, it terminates chain growing DNA chain, it terminates chain growth because another nucleotide cannot be growth because another nucleotide cannot be attached to it. Each of the four ddNTPs is attached to it. Each of the four ddNTPs is labeled with a different dye, which can later labeled with a different dye, which can later be detected using a special laser.be detected using a special laser.


CHAIN TERMINATIONCHAIN TERMINATION► The DNA polymerase The DNA polymerase

occasionally incorporates a occasionally incorporates a labeled dideoxynucleotide labeled dideoxynucleotide into a growing DNA strand. into a growing DNA strand. This doesn’t happen very This doesn’t happen very often, because the often, because the concentration of concentration of dideoxynucleotides is much dideoxynucleotides is much lower than the lower than the concentration of dNTPs in concentration of dNTPs in the reaction mixture. The the reaction mixture. The ratio of dNTPs to ddNTPs is ratio of dNTPs to ddNTPs is carefully balanced to get carefully balanced to get just the right number of just the right number of chain termination events.chain termination events.

Automated DNA Automated DNA SequencingSequencingPUTTING IT ALL TOGETHERPUTTING IT ALL TOGETHER►An actual sequencing reaction An actual sequencing reaction

mixture contains thousands of DNA mixture contains thousands of DNA template strands, which are all template strands, which are all being sequenced simultaneously. being sequenced simultaneously. Simply by chance, some annealed Simply by chance, some annealed primers will only be extended a primers will only be extended a few nucleotides before the chain few nucleotides before the chain extension is terminated by the extension is terminated by the addition of a ddNTP. addition of a ddNTP.

Automated DNA Automated DNA SequencingSequencingPUTTING IT ALL TOGETHERPUTTING IT ALL TOGETHER

►However, other primers will form a However, other primers will form a long chain of DNA before a ddNTP is long chain of DNA before a ddNTP is incorporated. Thus, there will be a incorporated. Thus, there will be a population of DNA strands in the population of DNA strands in the reaction, some very short, some very reaction, some very short, some very long, and every possible length in long, and every possible length in between. between.

Automated DNA Automated DNA SequencingSequencingPUTTING IT ALL TOGETHERPUTTING IT ALL TOGETHER

►To further increase the yield of To further increase the yield of sequenced strands, the sequencing sequenced strands, the sequencing reaction is performed in a thermal reaction is performed in a thermal cycler, which cycles through the cycler, which cycles through the heating and cooling steps dozens of heating and cooling steps dozens of times, in effect repeating the times, in effect repeating the sequencing reaction many times in sequencing reaction many times in one experiment. one experiment.


CAPILLARY CAPILLARY ELECTROPHORESISELECTROPHORESIS

► The newly synthesized The newly synthesized DNA strands, each DNA strands, each labeled with one of four labeled with one of four dyes, are now sorted by dyes, are now sorted by length using capillary length using capillary electrophoresis. First, electrophoresis. First, the reaction mixture is the reaction mixture is heated to keep the heated to keep the newly synthesized newly synthesized single strands from single strands from annealing with the annealing with the template DNA strand. template DNA strand. The dye-labeled single The dye-labeled single strands are loaded onto strands are loaded onto a tiny capillary tube a tiny capillary tube containing a viscous, containing a viscous, gel-like material.gel-like material.


CAPILLARY ELECTROPHORESISCAPILLARY ELECTROPHORESIS► An electrical current pulls An electrical current pulls

the negatively charged the negatively charged DNA strands through the DNA strands through the capillary. This tube is not capillary. This tube is not much thicker than a much thicker than a human hair and is 1 to 3 human hair and is 1 to 3 feet long, sufficient to feet long, sufficient to separate strands that separate strands that differ in length by only one differ in length by only one base. Because of the small base. Because of the small dimensions involved, dimensions involved, preparation of the capillary preparation of the capillary and loading of the sample and loading of the sample are computer controlled.are computer controlled.

Automated DNA Automated DNA SequencingSequencingCAPILLARY CAPILLARY

ELECTROPHORESISELECTROPHORESIS

► Shorter DNA strands Shorter DNA strands migrate through the gel migrate through the gel material more quickly, and material more quickly, and come out the bottom of come out the bottom of the capillary first, while the capillary first, while longer strands become longer strands become tangled in the gel material tangled in the gel material and take longer to emerge and take longer to emerge out the bottom.out the bottom.

Automated DNA Automated DNA SequencingSequencingCAPILLARY ELECTROPHORESISCAPILLARY ELECTROPHORESIS

► As the strands emerge out As the strands emerge out the bottom of the capillary the bottom of the capillary they pass through a laser they pass through a laser beam that excites the beam that excites the fluorescent dye attached fluorescent dye attached to the dideoxynucleotide to the dideoxynucleotide at the end of each strand. at the end of each strand. This causes the dye to This causes the dye to fluoresce, or glow, at a fluoresce, or glow, at a specific wavelength, or specific wavelength, or color. This color is then color. This color is then detected by a photocell, detected by a photocell, which feeds the which feeds the information to the information to the computer.computer.


COMPUTER ANALYSISCOMPUTER ANALYSIS► The computer displays the information received from the The computer displays the information received from the

photocell as an electropherogram, which is a tracing of signal photocell as an electropherogram, which is a tracing of signal received by the photodetector in each of the four wavelengths. received by the photodetector in each of the four wavelengths.

Automated DNA SequencingAutomated DNA Sequencing

In case of manual DNA sequencingIn case of manual DNA sequencing

manual DNA sequencingmanual DNA sequencing

The DNA sample is divided into The DNA sample is divided into four four separate sequencing reactionsseparate sequencing reactions containing the four standard containing the four standard deoxynucleotidesdeoxynucleotides (dATP, dGTP, dCTP (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. and dTTP) and the DNA polymerase.


manual DNA sequencingmanual DNA sequencing ► To each reaction is added To each reaction is added only one of the fouronly one of the four

dideoxynucleotidesdideoxynucleotides (ddATP, ddGTP, ddCTP, or (ddATP, ddGTP, ddCTP, or ddTTP). These dideoxynucleotides are the chain-ddTTP). These dideoxynucleotides are the chain-terminating nucleotides, lacking a 3'-terminating nucleotides, lacking a 3'-OHOH group group required for the formation of a required for the formation of a phosphodiester bondphosphodiester bond between two nucleotides during DNA strand between two nucleotides during DNA strand elongation. elongation.

► Various DNA fragments of varying length are Various DNA fragments of varying length are created in the created in the four separate sequencing four separate sequencing reactionreaction . .


manual DNA sequencingmanual DNA sequencing

► - The newly synthesized and labeled DNA fragments - The newly synthesized and labeled DNA fragments are heat are heat denatureddenatured, and separated by size (with a , and separated by size (with a resolution of just one nucleotide) resolution of just one nucleotide) by by gel electrophoresisgel electrophoresis on a denaturingon a denaturing polyacrylamidepolyacrylamide-urea-urea gel.gel.

► - Each of the four DNA synthesis reactions is - Each of the four DNA synthesis reactions is run in one of four individual lanesrun in one of four individual lanes (lanes A, T, (lanes A, T, G, C); G, C);

► the DNA bands are then visualized by the DNA bands are then visualized by autoradiographyautoradiography or UV lightor UV light, and the DNA , and the DNA sequence can be directly read off the sequence can be directly read off the X-ray filmX-ray film or or gel image. gel image.

► (A dark band in a lane indicates a DNA fragment (A dark band in a lane indicates a DNA fragment that is the result of chain termination after that is the result of chain termination after incorporation of one particular dideoxynucleotide incorporation of one particular dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). (ddATP, ddGTP, ddCTP, or ddTTP).

► - - The relative positions of the different bands The relative positions of the different bands among the four lanes are then used to read among the four lanes are then used to read (from bottom to top) the DNA sequence as (from bottom to top) the DNA sequence as indicated.indicated.

Automated VS manualAutomated VS manual

Automated DNA sequencing has the following advantages over Automated DNA sequencing has the following advantages over manual DNA sequencing manual DNA sequencing

► radioactivity is not used,radioactivity is not used,► gel processing after electrophoresis and gel processing after electrophoresis and

autoradiography are not needed, autoradiography are not needed, ► the tedious manual reading of gels is not required the tedious manual reading of gels is not required

as data are processed in a computer, as data are processed in a computer, ► the sequence data is directly fed into and stored in the sequence data is directly fed into and stored in

a computer, a computer, ► the separation of the same reaction products can the separation of the same reaction products can

be repeated to recheck the results in cases of doubt be repeated to recheck the results in cases of doubt since they can be stored for a long period of time, since they can be stored for a long period of time, and and

► it is extremely fast.it is extremely fast.

ApplicationApplication OfOf DNA SequencingDNA Sequencing Human Genome ProjectHuman Genome Project

Primary Application of DNA Sequencing is Human Genome Project Which is an Primary Application of DNA Sequencing is Human Genome Project Which is an international scientific research project with a primary goal to determine the international scientific research project with a primary goal to determine the sequence of chemical base pairs which make up DNA and to identify the sequence of chemical base pairs which make up DNA and to identify the approximately 25,000 genes of the human genome from both a physical and approximately 25,000 genes of the human genome from both a physical and functional standpoint.functional standpoint.

The work on interpretation of genome data is still in its initial stages. It is The work on interpretation of genome data is still in its initial stages. It is anticipated that detailed knowledge of the human genome will provide new anticipated that detailed knowledge of the human genome will provide new avenues for advances in avenues for advances in medicinemedicine and and biotechnologybiotechnology..

Diagnosis Diagnosis Example :Example :1-Noninvasive diagnosis of liver cirrhosis.1-Noninvasive diagnosis of liver cirrhosis.2-To Confirm Fungemia Due to 2-To Confirm Fungemia Due to Trichosporon dermatisTrichosporon dermatis in a Pediatric in a Pediatric

Patient. Patient.

ForensicForensic► Identify potential suspects whose DNA may match evidence left at Identify potential suspects whose DNA may match evidence left at

crime scenes crime scenes ► Exonerate persons wrongly accused of crimes Exonerate persons wrongly accused of crimes ► Identify crime and catastrophe victimsIdentify crime and catastrophe victims

DNA Sequencing Omar Alomair Homod Alherbi Fisal Alkulify.

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