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• 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
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
• DNA (negatively charged) travels from the Cathode end ( - ) to the Anode ( + ) end
• smaller fragments move through the gel faster than larger fragments
Pulsified Gel Electrophoresis
used to separate very large DNA fragments, including chromosomes
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)
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
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
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
- • 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
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)
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
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
- • 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)
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
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
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?
• 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
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
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
constructed directly from fragments of genomic DNA; includes introns & exons
derived from mRNA that’s converted to DNA by reverse transcriptase & then cloned; exons only
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
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.
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.
What is a plasmid?
A plasmid is a circular bacterial chromosome that replicates independently of the host chromosome.
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.
- • 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