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Restriction Endonucleases
• bacterial enzymes that recognize & destroy foreign DNA
• eg. EcoR I recognizes a specific sequence & cuts leaving fragments with 5′ over- hangs
• restriction endonuclease cleavage sites are palindromic
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How do restriction endonucleases differentiate endogenous (bacterial) DNA from foreign (eg. viral) DNA?
- bacterial DNA is methylated by endogenous enzymes, whereas foreign DNA is not
- this way bacterial enzymes can discriminate between endogenous & invading DNA
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Gel Electrophoresis
• DNA (negatively charged) travels from the Cathode end ( - ) to the Anode ( + ) end
• smaller fragments move through the gel faster than larger fragments
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Pulsified Gel Electrophoresis
used to separate very large DNA fragments, including chromosomes
size: 100-1000kb
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Steps of Southern Blotting
- 1. cleave genomic DNA with restriction enzymes
- 2. perform gel electrophoresis
- 3. transfer fragmented DNA from gel to nitrocellulose membrane
- 4. hybridize DNA of interest (eg. a gene) with labeled probe
- 5. expose membrane to film & develop
- (putting gel in NaOH denatures DNA, making it single stranded; can then add radioactive probe which will bind using complementary base pairing if fragment of interest is there)
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How to test for Sickle Cell using a Southern Blot:
• the mutation (GAG to GTG) is in a sequence recognized by a restriction endonuclease, so there is a loss of a restriction site
• when cut up then visualized on a southern blot the lost RE site leads to an alternately sized (larger) DNA fragment
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RFLPs (Restriction Fragment Length Polymorphisms)
- • individuals have polymorphic restriction endonuclease sites
- • individual differences can be examined by cleaving the DNA into restriction fragments
- • if a novel sized restriction fragment is closely associated with a gene containing a disease-producing mutation, the defective gene can be traced by the RFLP
*this technique requires no knowledge of the location or nature of the disease-producing mutation
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FISH (Fluorescence in situ Hybridization)
- • chromosomes are spread on microscopic slides which is exposed to a solution containing the labeled probe
- • careful treatment enables denaturation of the DNA without destroying the recognizable form of the chromosomes
- • chromosomes are gently denatured so they’ll hybridize to fluorescent labeled probe
- • used to check for the presence, absence or copy number of a chromosome
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Northern Blot
- • technique used to detect the size & amount of mRNA
- • RNA is separated according to size by electrophoresis through a denaturing gel & transferred to a solid support
- • RNA of interest is localized by hybridization with radiolabeled ssDNA or ssRNA
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Western Blot
detects the size & amount of a specific protein
• uses Antibodies specific for target proteins as probes (in contrast to Southern & Northern blots which use complimentary nucleic acid sequences)
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What does SDS-polyacrymalide gel do in the context of Western Blotting?
it’s anionic detergent that denatures proteins & gives them all the same charge, allowing for peptide separation according to size instead of charges
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DNA Band Shifts
determines if a protein interacts with a particular DNA fragment
• eg. if you want to determine if a certain transcription factor is present in a certain tissue
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DNA Footprinting
- • DNA is labeled only at a single end & protein is added to the labeled DNA sample
- • the reactions are then treated with a small amount of deoxyribonuclease I (DNase I) which digests DNA
- • regions of DNA covered by a bound protein will be protected from digestion
- • bands corresponding to cleavage at these points will be absent
- (a more precise determination of DNA-protein interactions)
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PCR (Polymerase Chain Reaction)
- 1. dsDNA is denaturation by heat (90° C)
- - ssDNA can potentially hybridize to any DNA molecule that contains a complementary sequence
2. Annealing: after cooling, 2 different oligonucleotides (primers) are annealed to their complementary sequences in the denatured DNA
- 3. Extension of annealed primers by Taq polymerase [heat stable]
- - dNTPS are also required
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Why can’t you amplify more than 5,000 base pairs using PCR?
• amplification is so large that we’re up against limitation of the PCR reaction
• polymerase is missing the clamp, it’s not processive enough
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Can you amplify a fragment of DNA using PCR if you do not know the DNA sequence of the regions that flank the fragment of interest?
No
• the sequence of the flanking regions must be known so that appropriate primers may be selected
• without probes that are complimentary to the flanking regions the DNA of interest will not be amplified
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RT-PCR
used to amplify a sequence of RNA
• a reverse transcriptase converts DNA into a ss-cDNA molecule, which can then be amplified using standard PCR techniques
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Single Strand Conformation Polymorphism (SSCP)
- • PCR products of interest are separated into single strands by chemical means (eg. formamide) & run on a polyacrylamide gel
- • given ssDNA migrates in a gel according to its conformation
- • if a mutation is present, the conformation is usually altered, therefore the band pattern will differ from the normal WT product
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Genomic Library
constructed directly from fragments of genomic DNA; includes introns & exons
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cDNA Library
derived from mRNA that’s converted to DNA by reverse transcriptase & then cloned; exons only
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The Sanger Method
- • enzymatic method of DNA sequencing that involves dideoxy-NTPs (ddNTPs) which lack both the 2' & 3' hydroxyl groups on the ribose
- • because the 3'-OH group on the ribose is required for chain elongation, incorporation of ddNTP's blocks chain elongation
- • might still be used for small fragments
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What are vectors?
Vectors are DNA molecules that replicate autonomously in a host cell. They are designed to accommodate the insertion of foreign gene sequences.
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After a vector is incorporated into a cell, how can recipient cells be sorted from non-recipient cells?
Vectors usually contain genes that confer resistance to particular drugs. Therefore, only recipient cells will be able to survive once the drug is added to the growth medium.
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What is a plasmid?
A plasmid is a circular bacterial chromosome that replicates independently of the host chromosome.
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A helicase is an enzyme that unwinds DNA. Why is a helicase not needed for the polymerase chain reaction (PCR)?
During PCR, the double-stranded DNA is denatured into single-stranded DNA by elevated temperatures, so the enzyme is not needed.
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Techniques Summarized
- • Southern Blot: separates DNA fragments
- • FISH: count chromosome #
- • Northern Blot: detects size & amount of mRNA
- • Western Blot: detects proteins
- • DNA Band Shift: determines whether a protein binds to DNA at a particular site
- • DNA Footprinting: to identify where a protein is sitting on DNA
- • PCR: DNA amplifier
- • RT-PCR: RNA amplifier by reverse transcribing it into cDNA
- • SSCP: uses ssDNA conformation to detect mutation
- The Sanger Method: DNA sequencing using ddNTPs
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