BIO2015: Lecture 4

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  • Created on: 12-05-14 11:41
what are the 2 methods of DNA sequencing?
1) Sanger dideoxynucleotide chain termination method (commonly used method) 2) chemical cleavage method (Maxam ad Gilbert)- not used nowadays
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what are the 2 methods of Sanger dideoxynucleotide DNA sequencing?
1) manual and automated
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what does the Sanger dideoxynucleotide method use?
it relies on the use of dideoxyribonucleoside triphosphates, derivatives of the normal deoxyribonucleoside triphosphates that lack the 3’-hydroxyl group.
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Purified DNA is synthesized in vitro in a mixture containing what?
single-stranded molecules of the DNA to be sequenced, the enzyme DNA polymerase, a short primer DNA to enable the polymerase to start DNA synthesis and the four deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP or A, C, G, and T)
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If a (blank) of one of these nucleotides is also present in the nucleotide mixture, it can become incorporated into a growing DNA chain
dideoxyribonucleotide analog
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what happens because this chain now lacks a 3’ OH group?
the addition of the next nucleotide is blocked, and the DNA chain terminates at that point.
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what will be the end product of this process?
DNAs of different lengths complementary to the template DNA that is being sequenced and terminating at each of the different A's. The exact lengths of the DNA synthesis products can then be used to determine the position of each A in the chain
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what is done to determine the complete sequence of a DNA fragment?
the double-stranded DNA is first separated into its single strands and one of the strands is used as the template for sequencing. Four different chain-terminating dideoxyribonucleoside triphosphates are used in four separate DNA synthesis reactions
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how are the products of these 4 reactions separated?
by electrophoresis in four parallel lanes of a polyacrylamide gel
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how are the newly synthesized fragments detected?
by a label (either radioactive or fluorescent) that has been incorporated either into the primer or into one of the deoxyribonucleoside triphosphates used to extend the DNA chain
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what do the bands in each lane of the polyacrylamide gel represent?
fragments that have terminated at a given nucleotide (e.g., A in the leftmost lane) but at different positions in the DNA.
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what are the nucleotides and primers labelled with?
S35 dNTP or P32 dNTP Or Fluorescent labelled dNTP
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which are used for automated and which for manual- radioactive or fluorescent labels?
radioactive labels= manual sequencing, fluorescent labels= automated sequencing
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what are the typical read lengths?
around 800 bases
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what do Fluorescently labeled ddNTPs allow?
allow all four termination reactions to be run in one lane - laser detection
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what improvements from the previous methods do Fluorescently labeled ddNTPs allow?
Big improvements to throughput, one tube reaction, no radioactivity, improved resolution of close bands
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However the maximum read-length of a sequence is about 1000 bp WHY?
This is due to convergence of the DNA bands
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what was the rate of sequencing in the 70's when it was developed and what is it now?
100 bp every 24 hours, now it is 1000,000 bp every 24 hours
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what are the 3 strategies for DNA sequencing of large cloned inserts?
1) directed cloning 2) primer walking 3) shotgun sequencing
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describe direct cloning
carefully preparing ordered overlapping fragments.
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describe primer walking
requires very fast and accurate analysis of sequence reads since each sequencing reaction uses information from the previous read- Entire DNA sequence generated by joining the individual strands in silico
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describe shotgun sequencing
takes maximum advantage of the speed and low cost of automated sequencing, but relies totally on software to assembly a jumble of sequence reads into a coherent and accurate contig.
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which three organisms did the institute for genomic research sequence?
1) Haemophilus influenzae 2) Methanococcus (genus) 3) Mycoplasma genitalium
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in primer walking where are all sequencing events initiated from?
all initiated from the same end of the template
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By a DNA sequencing reaction we can get up to how many bases of DNA sequence?
600-800 bases
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what does it mean that sequencing occurs in both directions?
Both the forward and reverse strand can be sequenced separately using specific primers
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what would happen in the case of 3kb DNA?
DNA has to be sequenced as a stretch of600-800 bases in five sequencing reactions. The resulting sequences has to be contig-assembled as shown in the picture above using software. eg. Sequencher, BIOEDIT, DNASIS, DNASTAR
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what are open reading frames?
Genes that encode for proteins comprise open reading frames (ORFs) within the DNA sequence- ORF will begin with an initiation codon (usually ATG) and end with a stop codon (TAA, TAG, TGA)
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how many reading frames does DNA have?
6 reading frames- genes can be present on both strands
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how large are most genes in terms of codons?
300-400- thus if you take a figure of 100 codons (including the start and stop codons) gives a good probability of finding ORF
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how often should you expect to see a codon?
every 100-200 bp by random - thus if you see a long stretch of DNA without stop codons likely that it contains an ORF
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does ORF scanning work well for eukaryotic or prokaryotic genomes?
works well for prokaryotic genomes. Genes are closely spaced, little intergenic DNA, small chance of mistaking a spurious ORF as a real gene
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why is ORF limited for eukaryotes?
genes are widely spaced (in humans 62% of the genome is intergenic - sometimes referred to as Junk DNA*), thus there is greater chance of identifying spurious ORFs. Also eukaryotic genes contain introns - so genes don’t appear as continuous ORFs in D
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what do BLAST based searches look for?
homologous genes/proteins- Homology means that the genes/proteins are related by evolution - i.e. shared ancestry
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what is the output from a BLAST search?
a list of homologous proteins and scores them according to how well the align.
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what is the Score (S) and E value?
S: a measure of the alignment similarity E value: is the expected number of chance alignments (random sequences) with a score of S or better
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what does COG stand for?
Clusters of Orthologous Groups (of proteins)
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what is proteomic?
coined by P. James in 1997- a sub-field of genomics concerned with characterising proteins via 2D gel electrophoresis and mass spectrometry.
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what is functional proteomics?
the identification of protein functions, activities or interactions at a global or organism-wide scale;
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what is expressional proteomics?
the analysis of global or organism-wide changes in protein expression
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what is structural proteomics?
the high throughput, or high volume expression and structure determination of proteins by X-ray, NMR or computer-based methods.
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what is the proteome?
the total protein content in a cell, tissue or organism.
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how large is the human proteome thought to be?
the human proteome is actually composed of more than 500,000 different proteins
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between the proteome and the genome, which is static and which is dynamic-condition dependent?
genome is static, proteome is dynamic conditions dependent
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which can be amplified the genome or the proteome?
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which has a high variability in amount- genome or proteome?
proteome, genome has no variability in amount
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what are the 2 methods of Sanger dideoxynucleotide DNA sequencing?


1) manual and automated

Card 3


what does the Sanger dideoxynucleotide method use?


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Card 4


Purified DNA is synthesized in vitro in a mixture containing what?


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Card 5


If a (blank) of one of these nucleotides is also present in the nucleotide mixture, it can become incorporated into a growing DNA chain


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