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DNA purification – very different from steps used to purify proteins...WHY?
due to differences in structure; first step is usually homogenization of cells & isolation of nuclei from which DNA is extracted.
PURIFICATION OF NUCLEIC ACIDS.. THE EXTRACTION MEDIUM. WHAT IS IT COMPOSED OF? (THE BASIC COMPONETS)
buffered salt solution + detergent, like SDS, that lyses nuclei, releasing DNA; solution viscosity rises as DNA is released; detergent also inhibits nuclease activity found in preparation
T OR F ?
solution viscosity rises as DNA is releasedinto it.
T OR F?
CHROSOMES ARE 55% PROTEINS.
WHEN GOING AFTER DNA WHAT 3 THINGS DO YOU HAVE TO REMOVE 1ST. (IN ORDER).
- LEAVES YOU WITH DNA.
Deproteinization usually accomplished by shaking extract with ________ OR ______________
phenol or phenol/chloroform mix
phenol or phenol/chloroform mix...WHAT DO THESE CHMS DO TO THE PROTEINS?
DEPROTEINIZATION/DENATURE...PROTEINS LOSS SOLUBILITY AND PRECIPITATE
Phenol & buffered saline are "immiscible",so one centrifuges to separate the phases..WHAT THE HELL DOES THAT MEAN?
Not forming a homogeneous mixture when added together. WHEN YOU SPIN DNA (& RNA) are in solution's upper aqueous phase; protein present as precipitate at boundary between phases
WHAT IS THE NAME OF THE LAYER THAT COULD BE FOUND INBETWEEN THE CHLOROFORM AND THE PHENOL WHEN DOING DNA EXTRACTION?
- BUFFY COAT.
- IT'S A BOUNDY LAYER BETWEEN THE TWOPHASES THAT IS COMPOSED OF PRECIPTATED PROTEINS. YOU CAN SIRENGE IT OUT AND REPEAT THE PROCESS TO GET MORE DNA/RNA SAMPLE.
IF YOU WANTED TO RUN A SPEC ON DNA/RNA OR PROTEINS WHAT LIGHT TYPE WOULD YOU USE?
UV SPECTRIUM. NOT COLOUR. SHORTER. 260-280nm. PRODUCES A RATIO. THE HIGHER RATIO IS BAD, GO BACK AND REDO PHENOL EXTRACTION.
4 M guanidine thiocyanate..WHERE WOULD YOU USE IT?
RNA EXTRACTION. USE IT TO HOMOGENIZE TISSUE WHEN GOING AFTER RNA SAMPLES.
4 M guanidine thiocyanate....WHAT TYPE OF AGENT IS IT?
WHAT IS THE MATRIX METHOD TO PURIFY DNA?
4 MAIN STEPS
- Cells are lysed in a solution that facilitates selective binding of DNA to the matrix.
- The lysate is applied to the matrix & contaminants are removed from the DNA by washing.
- Finally, the DNA is rinsed from the matrix with an elution buffer.
- These matrices are often set up in tiny columns inside centrifuge tubes.
of the same density
nucleic acids are separated on the basis of their buoyant density...NAME THE PROCEDURE
equilibrium (isopycnic) sedimentation
Mix highly concentrated heavy metal salt solution (CsCl or Cs2SO4) with DNA....WHAT PROCEDURE?
equilibrium (isopycnic) sedimentation, nucleic acids are separated on the basis of their buoyant density
In equilibrium (isopycnic) sedimentation, nucleic acids are separated on the basis of their buoyant density....NAME THE 2 BASIC STEPS
- A. Mix highly concentrated heavy metal salt solution (CsCl or Cs2SO4) with DNA
- B. Centrifuge with high force (speed) for extended period (2 - 3 days) to make continuous gradient (33-100,000g).
- THE CESIUM CREATES A GRADIENT.
WHAT CREATES THE DENSITY GRADIENT IN ISOPYCNIC SEDIMENTATION?
- CsCl....Caesium chloride
- Cs2SO4....Cesium Sulfate
HOW DO YOU ENTRACT THE DNA SEDIMENT FROM A equilibrium (isopycnic) sedimentation.
UNDER UV LIGHT, POKE THE LAYER YOU WANT WITH NEEDLE.
DNA molecules place themselves at the point in the gradient equaling their own buoyant density & they are then no longer subject to further movement...NAME THE PROCEDURE
equilibrium (isopycnic) sedimentation
equilibrium (isopycnic) sedimentation.. YOU HAVE A VERY NARROW BAND OF MOLECULES THAT HAVE THE EQUIVALENT DENSITY. WHAT CAN YOU USE TO SEPERATE THEM?
separate on basis of base composition or different isotopes of nitrogen (15N vs. 14N)
CsCl Equilibrium (ISOPYCNIC) Centrifugation.
REMEMBER THAT YOU HAVE ETHIUM BROMIDE IN YOUR EXTRACT AND YOU HAVE TO WASH IT OUT (ISOPROPENOL)(10X). WILL STICK TO YOUR DNA!
NAME A NEW DYE REPLACING ETHIUM BROMIDE.
HOW WOULD YOU BREAK DOWN A CHROSOME TO GET TO A PARTICULAR DNA FRAGMENT
- RESTRICTION ENZYME.
- MADE BY BACTERIA (Eco RI).
- IT'S A PARADROME.
- CAN DELIEVER VIA VIRUS.
INVERSE LOG OF BP SIZE. WHAT DOES THIS MEAN WHEN DOING A DNA (BP) GEL RUN?
SMALL ONES ARE EXPONETLY FASTER THAN LARGE ONES.
LETS SAY THAT YOU GET TWO CUTS FROM A DNA SAMPLE. HOW MANY CHAINS OF BP DO YOU REALLY HAVE?
- REMEMBER THAT DNA IS DOUBLE STRANDED!
NAME 3 BLOTS AND WHAT THEY GO AFTER
WHAT DOES PCR MEAN AND WHO DEVELOPED IT?
- Polymerase Chain Reaction (PCR)
- Developed by Kary Mullis (Cetus Corporation, 1983)
HOW LONG ARE PROBES FOR DNA?
WHAT PROCESS IS USED AMPLIFY DNA SAMPLES?
Polymerase Chain Reaction (PCR)
DO YOU USE BACTRERIA WITH PCR?
T OR F?
Many different PCR protocols have been developed for a multitude of different applications in which anywhere from one to a large population of related DNAs can be amplified
HOW CAN YOU USE PCR ON RNA?
by converting them to complementary DNAs using reverse transcriptase
PCR EXPONETIAL COPIES DNA SAMPLE.....WHAT DOES THAT MEAN?
- DOUBLE AFTER EACH RUN.
- START WITH 1...GET 2...RUN GET 4....RUN GET 8....RUN GET 16, ECT.
WHAT IS THE FIRST STEP FOR PCR?
Mix DNA sample with 4 deoxyribonucleotides & an aliquot of Taq polymerase. Also add a large excess of 2 short synthetic DNA fragments (oligonucleotides PRIMERS) that are complementary to DNA sequences at the 3' ends of the DNA region to be amplified.
Thermal Cycling...NAME THE 3 TEMPS AND WHAT HAPPENS AT EACH ONE.
- 95°C DENATURES AND SEPERATES DNA
- 60°C PRIMERS BIND TO BOTH DNA STRANDS.
- 72°C allows the thermophilic polymerase to add complementary nucleotides to the 3' end of the primers
As the polymerase extends the primers, it selectively copies the target DNAForms new complementary DNA strands
NAME THE 5 ITEMS IN THE TUBE BEFORE PCR STARTS.
- BUFFER....CONTAINS Mg
- TEMPLATE (TARGET DNA)
- TAQ POLYMERASE
- PRIMERS(OLIGONUCLEOTIDES)(REV & FWRD)
- ddDNA (A,T,C,G)
AKNEELING OCCURS AT WHAT TEMP IN PCR?
HOW DO YOU CHANGE THE STRIGENCY OF A PCR RUN?
65C INSTEAD OF 60C. TEMP ALLOWS PRIMERS TO FIND PERFECT MATCH.
WHAT IS THE OPTIMIAL TEMP FOR POLYMERASE?
WHAT IS THE TIME FRAME FOR EACH OF THE THERMAL CYCLING?
30 SECONDS EACH
One selects regions of genome for amplification that are highly polymorphic (i.e., vary at high frequency within population) - thus, no 2 individuals will have same-sized DNA fragments.
HOW IS THIS USED?
GENETIC FINGERPRINTING, CRIME FIGHTING.
it took much longer to determine DNA sequences than protein sequences - why?
- 1.Unlike DNA molecules, polypeptides come in defined & manageable lengths.
- 2. CLEAVING POLYPEPTIDES GAVE OVERLAPS
first polypeptide amino acid sequence...WHO, WHAT & WHEN?
- BOVIN insulin
2 original sequencing techniques
- Developed almost simultaneously
- Sanger & Coulson Cambridge (BETTER)
- Maxam & Gilbert. Harvard
- The Sanger-Coulson method used an enzymatic approach
- The Maxam-Gilbert method used a chemical (DEGREDATION) approach
MISSING A HYDROXYL GROUP IN THE 3" POSITION
WHAT DID SANGER'S METHOD EVOLVE INTO?
Dideoxy (Sanger-Coulson or chain termination) technique
Dideoxy (Sanger-Coulson or chain termination) technique..
- Begin with population of identical DNA fragments (up to ~500 bp in length) - obtained by treating DNA with restriction enzyme.
- Divide preparation into 4 samples, denature each into single strands & treat them slightly differently.
- Incubate each preparation with short, radiolabeled oligonucleotide complementary to the 3' end of one of the single-stranded fragments.
- READ RESULTS
WHY USE ddATP (ddGTP,ddCTP OR ddTTP)
CAN'T GO PAST THE 3' WHEN DIDEOXY LOCKS IN PLACE "TERMINATION"
WHAT GEL TYPE DO YOU USE WITH SANGER METHOD?
POLYACRMID. JUST LIKE PROTEINS. TIGHER BANDS BECAUSE DIFFERENCE OF JUST 1 BP.
READ THE SEQUENCES:
WHATS INSIDE THE SANGER METHOD REACTION MIXTURE? 5 ITEMS
- 1. OLIGONUCLEOTIDE PRIMER (ONLY ONE)
- 2. DNA polymerase
- (most often bacterial DNA polymerase I)
- 3. ALL 4 deoxyribonucleoside triphosphate precursors (dNTPs)
- 4. low concentration of a modified precursor dideoxyribonucleoside triphosphate (ddNTP)
- 5. A different ddNTP (ddATP, ddTTP, ddCTP, ddGTP) is added to each of the four samples
ddNTPs lack ________ AT ___ & _____ positions —> once a ddNTP is incorporated onto chain end, the chain is terminated, since there is no 3'-OH group; polymerase cannot add any more nucleotides
oxygen at 2' & 3' positions
Since ddNTP is present at much lower concentration in reaction mixture than corresponding dNTP,WHAT HAPPENS?
Incorporation of ddNTP is infrequent & random.
It may be incorporated near beginning, middle or end of a chain —> get fragments of different size
Successive labeled bands correspond to fragments with common____ ____ but increasing length of ___ _______ & the __ _______ nucleotide in each band is known by virtue of lane in which it lies
- 1 NECLEOTIDE
- 3' TERMINAL
Identify each of the introns in the DNA that interrupts coding; each intron-exon boundary begins & ends with precise nucleotide sequence —> so introns can usually be easily identified
DEFINE intron-exon boundary
DNA restriction fragments are usually labeled by _________ labeled precursors instead of radioactively labeled precursors
Next Generation Sequencing (NGS)...WHAT'S CHANGED FROM THE SANGER METHOD?
- •No need for cloning in yeast or bacteria
- like HGP.
- •Fragments directly from genome
- •Still uses PCR process, but…..
- –No premature strand termination...NO dd's
- –No gel electrophoresis..FLOURENCE TAGS. CMPTR READS.
Three new instruments rapidly sequence DNA today
(Post-Sanger DNA Sequencing)...NAME 3
- pyro sequencing
- SOLEXA sequencing
- SOLiD technology
Most Current techniques do not requires amplified DNA or enzymatic activity....BASICALLY WHAT'S DONE?
DNA PULLED THRU NANOPORE, READS SEQUENCE.
Example of DNA cloning using Bacterial Plasmids