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Transcript of DNA Sequencing
By Meeki Lad
AP Biology Block 1
dna, or deoxyribonucleic acid, is a nucleic acid that carries the cell's genetic information. It physically takes the form of a double helix
This is one of the 2 deoxyribose sugar-phosphate strands.
The strands run antiparallel. One runs 5'->3', the other runs 3'->5'.
there are four nucleotides that make up DNA, each with its own nitrogenous base. Nitrogenous Bases form hydrogen bonds between each other (A:T and G:C)
Helicase unwinds the parental double helix as single-strand binding proteins stabilize the unwound bands and topoisomerase stabilizes the ends of the replication bubble. A primer of RNA is added by Primase.
The leading strand is synthesized continuously bu DNA pol. III in the 5'->3' direction. On the complementary strand, separate okazaki fragments are synthesized by a plethora of primers.
The OKAZAKI FRAGMENTS ARE BONDED TOGETHER by LIGASE after dna pol. 1 replaces the rna primer with dna.
DNA is successfully replicated and can be stored in two daughter cells. this is pivotal as DNA essentially determined all cellular functioning through the process of transcription and translation.
DNA Sequencing: The Basics of DNA
First methods for sequencing developed in the 1970s. Only a few bases per year could be sequenced
In 1990, the Human Genome Project began; at the time, sequencing was expensive and only a few labs had sequenced the seemingly unreachable 100,000 nucleotide limit
Today, advanced methods, namely chain termination, has allowed for the sequencing of several species.
heat is added to break apart the dna strands
Chain Termination, Step 2:
Replication With Dideoxynucleotides
Deoxinucleoetides lack a 3' hydroxyl group and thus no other nucleotides can bond to these compounds.
When deoxinucleotides are added to the reaction mix, dna fragments of various lengths are synthesized:
Chain Termination, Stage 3:
Gel Electrophoresis and Fluorescence
Because the fragments vary in size, gel electrophoresis can be used to separate strands in order of length. By coloring the deoxinucleotide (e.g., a blue guanine), the distance between those nucleotides in the dna sequence can be estimated.
If every deoxinucleotide is augmented so that it fluoresces, a continuous dna sequence of up to 900 nucleotides can be determined by the electrophoresis
Chain Termination, Step 4:
The location of each colored band on the electrophoresis tray can be scanned by a machine to determine the nucleotide sequence
Alternative Method to Chain Termination: Shotgun Sequencing
By this method, a large DNA segments is fragmented and inserted into plasmids (circular DNA molecules found in bacteria). The bacteria grow; the DNA multiplies. The inserts are sequenced and overlapping sequences are used to derive a final sequence of the original DNA.
dna sequencing is, as its name implies, the process of determining the nitrogenous base sequence of a segment of DNA. Once coded, the sequence can be paired with the cellular response it espouses, and later be used for recognition purposes.
DNA Sequencing: The Process
DNA Sequencing: Its Application
Chain Termination, Step 1: Isolation and Amplification
the template DNA is placed in a reaction mixture with free nucleotides and dna polymerase.
Experiment Name: Viral discovery and sequence recovery using dna microarrays
authors: David Wang, Anatoly Urisman, Yu-Tsueng Liu, Michael Springer, Thomas G Ksiazek, Dean D Erdman, Elaine R Mardis, Matthew Hickenbotham,Vincent Magrini, James Eldred, J. Phillipe Latreille, Richard K Wilson, Don Ganem, Joseph L DeRisi
Where: UC San Francisco
Modern methods of viral recognition and discovery are limited; a new approach is needed. in this experiment, the tested approach is the use of a DNA microarray-based platform for novel virus recognition.
Goal: Be able to detect an unknown virus in and SARS patient using a microarray platform.
Creating the Microarray
Initial microarray: consisted of 1600 oligonucleotides representing 140 viruses
second generation microarray: consisted of 10000+ 70mer oligonucleotides, with the 10 most common oligonucleotides of each of 1000 viruses
Creating 4 Tested Samples
Vero cells from a patient suffering from SARS were taken and cultured in four solutions. The DNA from the SARS cell assimilated dna in the culture.
1. Control Culture (No Virus)
2. Viral Culture (UNKNOWN Virus)
3. HeLa Culture (HeLa RNA)
Eight oligonucleotides hybridized more frequently with Sample 2 than all the other samples
Two families of viruses hybridized: coronaviruses and astroviruses
Since coronavirus and astrovirus oligonucleotides hybridized, the virus culture SEEMS to have contained some of both
5 of the 8 oligonucleotides had a common sequence motif of 33 nucleotides; they were hybridizing redundantly for the complementary sequence.
The remaining 3 were all found in regions of the ORF1AB polyprotein common to all coronaviruses
The 5 redundant oligo sequences are common to the 3' UTR region of all astroviruses and avian coronaviruses (e.g, avian IVB). The other oligo sequences imply that the virus is of the corona family. However, since there are multiple oligo sequences of DIFFERENT viruses, the coronavirus appears to be a novel one.
Further Analysis: BLAST Alignment
Deductions From the Previous Step:
The virus is a coronavirus containing a special 3' UTR region.
Three other oligo sequences - IVB, Human Coronavirus 229E, and Bovine Coronavirus - were shown to hybridize as well.
what do these three sequences have in common?
This segment was found in the unknown virus.
This segment was also found in the unknown virus
This segment should also be found in the unknown virus
89% homology to MHV, a mammalian coronavirus
Final Confirmation: Sequencing the 3' UTR Motif
THE 3' UTR Spot of the microarray was scraped for the hybridized viral sequence, which was then amplified via pcr and cloned. once sequenced, blast analysis revealed 33% homology to MHV over 157 amino acids. Shotgun Sequencing confirmed this.
The virus is a novel coronavirus variant of MHV. This can be deduced by the homologies in the ORF1AB polyprotein to that viral amino acid sequence as well as homologies in the 3' UTR region. It varies from normal MHV in that it possesses the 3' UTR motif of astroviruses/avian coronoviruses and in that the ORF1AB sequence is slightly different.
ALL Methods Used Throughout This Experiment and their Relevance to DNA Sequencing
DNA Sequencing Core.
University of Michigan, 23 Dec. 2013. Web. 31 Dec. 2013.
National Institutes of Health, 27 Dec. 2011. Web. 31 Dec. 2013.
Wang D, Urisman A, Liu Y-T, Springer M, Ksiazek TG, et al. (2003) Viral Discovery and Sequence Recovery Using DNA Microarrays. PLoS Biol 1(2): e2. doi:10.1371/journal.pbio.0000002
Nucleic Acid Isolation.
Incubation in NucliSens lysis buffer of the samples followed by transfer to extraction cartridge.
Like in Chain Termination, DNA must first be isolated.
Mixing of nucleic acid in samples with primers in culture to allow for DNA replication.
Like in Chain Termination, DNA must undergo the process of forced replication.
Polymerase Chain Reaction.
A process of amplification by which targeted primers aligned to hybridizing oligonucleotides are combined with thermocycling DNA.
In chain termination, PCR primer amplification is the most commonly used procedure.
Microarray Creation and Usage.
The creation of various oligonucleotide sequences that then fill a small chip which is placed in the presence of DNA sequences and presumably fluoresces in the presence of hybridization with matching complementary sequences.
The creation of the oligonucleotide complements for the included viruses is made possible by GenBank, a collection of DNA Sequencing results funded by the Human Genome Project.
BLAST and ClustalX Alignment.
Matches DNA sequences of a segment to those in the sequence database of another segment. Also used for amino acid matches.
Aligment takes advantage of sequence databases and requires the sequencing of new strands.
Sequence Recovery From Microarray.
Scraping of hybridized microarray spot followed by immediate PCR amplification, cloning, and sequencing.
The entire process of Chain Termination is pivotal here as it is used to determine the sequence of several viral DNA segments.
The aforementioned process of sequencing several small segments of DNA and finding overlapping zones to determine a full sequence.
This is a type of DNA sequencing.
general importance of dna sequencing:
dna sequencing is ultimately pivotal to this experiment as it allows for:
the creation of the microarray
The determination of what sequences are being redundantly probed
evaluation of similarities with currently sequenced diseases as well as unsequenced ones
the sequencing of novel viruses.