What is the PE solution?
a proprietary formula of ethanol-salt
What is the E value (E)?
Expectation value. Is the number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance.
What is the BLAST cutoff for homology of protein based searches?
E= 10^-3 sequence identity= 25%
Why is the sequence broken into fragments "words"
-improves the overall speed of searches -retains good sensitivity -allows word hits to extend in either direction to generate an alignment with a score exceeding the threshold of "S"
What is the purpose of the PE solution?
PE washes away trace amounts of primers, salts, and dNTPs of the PCR
What is the BLAST cutoff for homology of nucleotide based searches?
E= 10^-6 Sequence identity= 70%
How does the DNA stay bound to the silica membrane
the ethanol in the PE solution prevents the DNA from washing away
What is the different rate of movement of DNA of different sizes?
shorter or more compact DNA will move faster than longer and more convoluted ones
What is the purpose of the PB solution?
allows >100 bp of DNA to bind to the membrane
What is the charge of DNA?
What is contained in the QIAGEN QIAquick Kit?
1. spin column with silica gel membrane 2. PB solution (binding solution) 3. PE solution (ethanol-salt) 4. EB solution (elution buffer)
what molecule did we use to increase the density of the DNA sample?
What are the steps to the purification procedure?
1. DNA binding 2. DNA washing 3. DNA elution
When database gets bigger, what happens to E-value?
Bioinformatics resources are provided by a number of sources including:
NCBI (by the NIH and national library of medicine European Molecular Biology Lab DNA Data Bank of Japan
What is the loading buffer made up of?
-Tris buffer, EDTA, and 3 negatively charged indicator dyes -Sucrose,glycerol, or Ficoll ( a polysaccharide) are used to increase the density of the DNA sample so it sediments at the bottom of the loading well
What occurs in the first step of purification?
1. PB DNA binding: DNA binds in the presence of high salt and chaotropic ions (disrupt water structure)
Compares a nucleotide query sequence translated in all reading frames against a protein sequence database. You could use this option to find potential translation products of an unknown nucleotide sequence (nucleotide all frames-->prot)
When can regions be highly similar without being homologous?
When they are regions of low complexity. Homologous sequences are not always highly similar
What is the running buffer?
-The buffer in which the gel is made and used for electrophoresis -Tris-acetate-EDTA (TAE buffer)
What is represented by the score (S)?
the quality of the alignment
What is BLAST based on?
based on Smith Waterman algorithm. heuristic approach
What is the loading buffer?
added with DNA to monitor the electrophoresis and sediment the sample in the gel well
What's a scoring matrix? What is used for AA alignments? What is used for DNA pairs?
-substitution matrices used for AA alignments. Each residue substitution is given a score -Simpler unitary matrix for DNA pairs (+1 match, -2 mismatch)
How much DNA can bind to the column?
Up to 10 micrograms
What is the direction of electrophoresis?
-anode to cathode - positive to negative -anode (positive side) -cathode (negative side)
What makes dideoxyribonucleotides different from normal nucleotides?
The 3' OH group is changed to an H so that no more nucleotides can be added
compares a protein sequence against a nucleotide sequence database dynamically translated in all reading frames (prot Q-> nucleotide all frames)
What is the silica membrane column?
-column that reactions will take place in -easily subjected to quick centrifugation -fits over most 1.7 ml microcentrifuge tubes -high DNA binding
What is the PB solution?
(binding solution) made up of: -high salt concentration -quanidine hydrochloride (a chaotrope, disorders) -isopropanol (proprietary)
What is the score (S)?
-represents quality of each pair-wise alignment as a score and scores are ranked -scoring matries calculate the score of the alignment base by base (DNA) or amino acid by amino acid (protein) -The alignment score will be the sum of the scores for each position
compares an AA query sequence against a protein sequence database
What is the purpose of electrophoresis?
to separate macromolecules based on their charge and size
What are the statistical features of BLAST?
-discriminates between real and artifactual matches -uses an estimate of probability that the match might occur by chance -produces scores (S) and e-values (E)
What is the EB solution?
elution buffer. 10 mM Tris-Cl, pH 8.5
What is the blast algorithm?
-scoring of matches done using scoring marries -sequences are split into "words" (n=3) -Blast algorithm extends the initial "seed" hit into an HSP. (high scoring segment pair= local optimal alignment)
When does sequence data become ambiguous on the DNA sequence chromatogram?
after 750-800 base pairs
What is the DNA sequence read from?
a spectrogram (chromatogram)
Which indicator dye shows 50 bp?
Orange G, orange color
What are the different methods of quantifying DNA?
1. measuring absorbance at ultraviolet wavelengths (260 nm) 2. agarose gel electrophoresis with ethidium bromide (EtBr)
What occurs in the second step of DNA purification?
2. PE DNA washing: DNA is washed with ethanol salt solution that removes trace amounts of reaction salts and primers but keeps DNA bound to silica membrane
How does the DNA molecule move through the gel matrix?
An electric field is applied to move the negatively charged DNA
Genbank,doubles its data approximately every
18 months. (Genbank= NCBI database)
What is the direction of movement of the DNA strands by the label detector?
left to right: longest to shortest (shortest first)
What occurs in the final step of DNA purification?
The EB solution elutes the DNA. Low ionic strength, alkaline pH,DNA redissolves and elutes
compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database
What is the gel used for electrophoresis reaction?
1.5% (1.5g/100 ml) agarose gel
What is represented by the E value (E)?
The significance of each alignment. Lower E value means the results are more significant
What is the role of EtBr in agarose gel electrophoresis?
EtBr intercalates between the bases of double stranded DNA and increases fluorescence
When alignments get longer, what happens to E-value?
Which indicator dye shows 300 bp?
Bromophenol blue, blue color
Which indicator dye shows 4000bp?
Xylene cyanole, gives off a teal color
Compares a nucleotide query sequence against a nucleotide sequence database
What does a low E value suggest?
-E-value suggests that sequences are homologous. **difficult to show non-homology
What does BLAST stand for?
Basic Local Alignment Search tool
What are the steps of using BLAST?
1. submit query 2. BLAST webpage 3. Request results 4. Blast server 5. Sequence data 6. Return formated results 7. Blast webpage 8. Displayed results
What is the purpose of the EB?
The elution buffer elutes the DNA from the membrane into the solution