BIO2015: Lecture 1

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  • Created by: LMoney
  • Created on: 11-05-14 19:37
DNA must be extracted as high purity for (blank)
downstream applications
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what ratio indicates DNA purity?
A260/A280
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what value of this ratio indicates a high purity of DNA?
1.7-2.0
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what must be done to prepare a recombinant DNA molecule?
the DNA that is to be cloned/manipulated, must be cut at specific points and then joined together in controlled manner
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Cutting and joining DNA molecule are two ‘famous’ examples of DNA manipulation techniques, what are some other examples of DNA manipulation techniques?
DNA molecule can be shortened lengthened, copied into RNA or into new DNA molecule- can also be modified by addition or removal of specific chemical groups
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Almost all DNA manipulative techniques use what?
purified enzymes
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what are some examples of what these enzymes do in their original cell location?
DNA replication, transcription, breakdown of foreign/unwanted DNA, repair of mutated DNA and recombination between different DNA molecules
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what are the 5 broad classes of DNA manipulative enzymes?
Nucleases, ligases, polymerases, modifying enzymes, topoisomerases
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what do nucleases do?
enzymes that cut, shorten or degrade nucleic acid molecules
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what do ligases do?
join nucleic acid molecules together
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what do polymerases do?
make copies of molecules
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what do modifying enzymes do?
remove or add chemical groups
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what do topoisomerases do?
introduce/remove supercoils from covalently closed circularly DNA
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how exactly do nucleases cut, shorten or degrade nucleic acid molecules?
break phosphodiester bonds that link 1 nucleotide to the next
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what are the 2 types of nucleases?
1) exonucleases 2) endonucleases
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how do exonucleases act?
remove nucleotide one at a time from the end of a DNA molecule
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how do endonucleases act?
break internal phosphodiester bonds with DNA molecule
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what are some examples of endonucleases?
S1 nuclease, Mung Bean Nuclease, RNAse A, Restriction enzyme
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what are the 2 types of RNAses?
RNAse A and RNAse H
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describe RNAse A
endoribonuclease that specifically attacks SS RNA and not DNA
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describe RNAse H
endoribonuclease that digests the RNA of an RNA-DNA hybrid
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what specifically are the actions of ligases
1) Repairs single stranded (**)-break in one of the strands of a double stranded (ds) molecule 2) • Also joins together individual DNA molecule or the two cohesive ends of the same molecule
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what is the mode of action of a ligase?
catalyzes formation of a phosphodiester bond between adjacent 3’-OH and 5’-P termini in DNA
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which is more efficient: ligation of complementary or blunt ends?
complementary- this is because compatible sticky ends can base pair with one another by hydrogen bonding- forms relatively stable structure for enzyme to work on
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what exactly are polymerases?
The enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template
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what do most polymerases need?
a template
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what are the 4 types of DNA polymerases used in molecular biology?
1) DNA polymerase I (usually from E.coli & T4 phage) 2) Klenow fragment DNA polymerase 3) reverse transcriptase (RNA-dependent DNA polymerase) 4) TaqDNA polymerase (PCR enzyme)
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what functions does DNA polymerase have?
5’ to 3’ polymerase; 5’ to 3’ exonuclease & 3’ to 5’ exonuclease (enzyme with dual functions- DNA polymerization & DNA degradation)
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what are its common uses?
commonly used in Nick translation & probe preparation, repairing of DNA fragments, producing a blunt end DNA from a sticky ends DNA
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what are the functions of Klenow fragment DNA polymerase?
has 5’ to 3’ polymerase & 3’ to 5’ exonuclease activities, no 5’ to 3’ exonucleaseactivity, can only synthesize complementary DNA strand on a single stranded template
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where is Klenow fragment DNA polymerase commonly used?
1) sanger dideoxy sequencing and synthesis of second strand cDNA in cDNA cloning 2) filling in the 3’ recessed termini created by digestion of DNA with RE & labeling the termini of DNA fragment- end filling reaction
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what are the functions of reverse transcriptase (RNA-dependent DNA polymerase)?
has 5’ to 3’ polymerase, 5’ to 3’ riboexonuclease and 3’ to 5’ exoribonucleases activities
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what are the common uses of reverse transcriptase?
1) synthesis of cDNA for cloning 2) labelling the termini of DNA fragments with protruding 5’ ends (filling reaction)
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what are the functions of TaqDNA polymerase?
only has 5’ to 3’ polymerase activity only no 3’ to 5’ exonuclease (no proofreading activity)
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what are Taqpolymerase's common uses?
1) widely used in PCR reaction (requires specific primers)-latest version of Taq has proofreading activities with even higher polymerization capabilities
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what are 4 examples of DNA modifying enzymes?
1) alkaline phosphatase (AP) 2) polynucleotide kinase 3) terminal deoxynucleotide transferase 4) DNA methylase (dam & dcm)
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what are alkaline phosphatases (APs)?
removes phosphate group present at the 5’ terminus of DNA molecule- use: prevents recircularization of plasmid during cloning work
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what are polynucleotide kinases?
adding phosphate groups on to free 5’ termini (reverse of AP)
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what are terminal deoxynucleotide transferases?
add 1 or more deoxynucleotides onto the 3’ terminus of DNA molecule uses: 3’ tailing reaction
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what are DNA methylases (dam & dcm)?
transfer of methyl group to internal A or C residues
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what does a class 1 restriction endonuclease do?
recognize some specific sequence like all other Res enzymes, but are not particularly useful in gene manipulation since cleavage site is non-specific- have methylase activity
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what does a class 2 restriction endonuclease do?
Mg2+ dependent with specific recognition site- very useful for DNA manipulation work
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what does a class 3 restriction endonuclease do?
have nuclease & methylase activity- recognition sites are not symmetrical
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how do class 2 restriction enzymes cut DNA?
they cut both strands of DS DNA with a (normally symmetrical) recognition sequence
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how is this achieved on a chemical level?
hydrolyze sugar phosphate backbone to give 5’-phosphate on one side and 3’- OH on the other side- yield blunt or sticky ends (5’ to 3’ overhang)
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who won a nobel prize for the discovery of these enzymes?
W Arber, H Smith, and D Nathans in 1978
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what is an isoschizomer?
different REs (from different sources) that can recognize same restriction site
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what is a Palindromic sequence in DNA?
reads alike backward and forward
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how are restriction enzymes named?
Species name of the organism that produces the enzyme: Escherichia coli = Eco, Haemophilius influenza= Hin, Strain will be written after species name e.g. EcoR
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what happens if the strain has different restriction or modification systems?
these will be identified by Roman Numerals: e.g. EcoRI
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what is the purpose of Gel electrophoresis?
Resolves DNA fragments of different sizes
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what are the 2 ways that visualization of restriction fragments separated by gel electrophoresis can be accomplished?
1) Using staining with the fluorescent dye ethidium bromide 2) Using radioactive labeling followed by autoradiography
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what kind of gel electrophoresis separates large DNA molecules?
Pulsed-field gel electrophoresis
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what ratio indicates DNA purity?

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A260/A280

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what value of this ratio indicates a high purity of DNA?

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what must be done to prepare a recombinant DNA molecule?

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Cutting and joining DNA molecule are two ‘famous’ examples of DNA manipulation techniques, what are some other examples of DNA manipulation techniques?

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