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Base analogs
chemicals with structures similar to any of the four standard bases
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5-bromouracil
base analog for thymine which can mispair with guanine leading to a transition mutation
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2-aminopurine (2AP)
base analog of adenine which can mispair with cytosine -> transition mutation
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EMS/Alkylation
guanine->O6-Ethylguanine: pairs with thymine -> transition mutation
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Nitrous acid/Deamination
cytosine->Uracil: pairs with adenine -> transition mutation
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Hydroxylamine/Hydroxylation
cytosine->Hydroxylaminocytosine: pairs with adenine -> transition mutation
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oxidative radicals/reactions
reactive forms of oxygen that damage DNA and induce mutations via chemical change
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intercalating agents
molecules which insert into DNA in place of nitrogenous bases causing insertions and deletions, e.g. proflavin and acridine orange
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Hermann Muller
1927: showed mutations in fruit flies inducible by radiation
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x-rays, gamma rays, cosmic rays
alter base structure, break phosphodiester bonds, and even cause double-strand breaks
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UV light
less energy, but still mutagenic
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pyrimidine dimers
can be caused by UV light; create covalent bonds between bases which block replication
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SOS system
eukaryotic system of eta polymerase that can bypass pyrimidine dimers
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Mismatch repair
Replication errors, including mispaired bases and strand slippage
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Direct repair
Pyrimidine dimers; other specific types of alterations
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Base excision
Abnormal bases, modified bases, and pyrimidine dimers
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Nucleotide excision
DNA damage that distors the double helix, including abnormal bases, modified bases, and pyrimidine dimers
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Mismatch repair Mechanism
- Detects 3D distortion
- cuts out distored part with exonuclease
- fill in using original strand as template with DNA polymerase
- repairs nicks with DNA ligase
- Methylation at GATC sequence on old strand for differentiation
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exonuclease
removes nucleotides usually at end of DNA strand
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DNA polymerase
replaces nucleotides
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DNA ligase
seals nick in sugar-phosphate backbone
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Direct Repair Mechanism
- Converts modified nucleotides to original form
- e.g. O6-Methyltransferase removes methyl group restoring base to guanine
- e.g. photolyase uses light to break covalent bonds that link pyrimidine dimers
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photolyase
an enzyme that uses light energy to break covalent bonds in pyrimidine dimers
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Base-excision repair mechanism
- Excises modified bases and then replaces entire nucleotide
- DNA glycosylase recognizes and removes damaged base
- AP endonuclease cleaves phosphodiester bond on 5' site and removes sugar
- DNA polymerase adds new nucleotide to 3'
- DNA ligase fixes nick in sugar-phosphate backbone
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AP endonuclease
removes nucleotide usually in middle of DNA strand
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Nucleotide-excision repair mechanism
- Enzyme complex recognizes 3D distortion
- DNA strand is separated and stabilized with binding proteins
- An enzyme cleaves the strand on both sides of the damage
- Part of damage strand is removed
- Gap filled by DNA polymerase
- Sealed by DNA ligase
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Homologous recombination
A type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.
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Interstrand cross-link
Two DNA strands are connected thru covalent bonds. Not much know about this.
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Common Mechanisms for Nucleotide removal
- Detection
- Excision
- Polymerization
- Ligation
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Detection
Damaged section of DNA is recognized
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Excision
DNA repair endonucleases nick phosphodiester backbone on one or both sides of the DNA damage and one or more nucleotides are removed
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Polymerization
DNA polymerase addes nucleotides to the newly exposed 3'-OH group by using the other strand as a template and replacing the damaged nucleotides
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Ligation
DNA ligase seals the nices in the sugar-phoshate backbone
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Differences in Mechanisms for Nucleotide removal
How detection and excision are accomplished
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xeroderma pigmentosum
autosomal recessive condition caused by nonfunctional repair mechanism for pyrimidine dimers
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1973, Cohen and Boyer at UCSF
Created first recombinant DNA molecule
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Recombinant DNA technology
Set of molecular techniques for locating, isolating, altering, and studying DNA segments
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Stemps requiring Recombinant DNA techniques
- Find gene
- Separate gene
- Make copies of gene
- Insert gene into plasmid without degredation
- Induce bacteria to take up plasmid
- Select bacteria that take up plasmid
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Restriction enzymes/endonucleses
- Enzymes that recognize and make double-strand cuts in DNA at specific nucleotide sequences
- Produced naturally by bacteria to defend against viruses
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Type I and III Restriction enzymes
Cut outside recognition sequence
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Type II restriction enzyme
- Cuts within recognition sequence
- Used in molecular genetic work
- Names indicate original bacteria
- More than 800 isolated
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Characteristics of restriction enzymes
- Palindromic recognition sequence
- Fragment end either cohesive or blunt
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Cohesive end restriction enzyme
Staggered cut -> sticky ends
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Restriction Digest
Reaction of mixture of DNA, buffer, restriction enzyme, water heated at about 37 C
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Electrophoresis
Standard technique for separating molecules on basis of size/electrical charge
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agarose
polysaccharide isolated from seaweed
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Viewing DNA fragments using electrophoresis
DNA fragments move to positive pole with smaller fragments moving faster
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probe
fluorescent or radioactive DNA or RNA fragment complementary to sequence of interest
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Southern blotting
Transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by radioactive probe
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Northern blotting
- A technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.
- Size of mRNA molecule
- Relative abundance of mRNA
- Tissue in which MRNA is transcribed
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Western Blotting
- Transfer of proteins from gel to a membrane
- Probe is usually an antibody
- Determine size of protein
- Pattern of protein's expression
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Gene cloning
create identical copies of a piece of DNA
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Cloning vector
DNA molecule into which a foreign DNA fragment can be inserted for introduction into and replication in a cell
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Characteristics of an effective cloning vector
- Origin of replication
- selectable marker
- one or more unique restriction sites where DNA can be inserted
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Types of cloning vectors
- Plasmid, e.g. pUC19
- Bacteriophage
- Cosmid
- BAC
- YAC
- Retroviral vectors
- Transposons
- Expression
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Transformation
The capacity of bacterial cells to take up DNA from the environment
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Cosmid
Plasmids that are packaged into empty viral protein coats and transferred to bacteria by viral infection
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BAC
Originally contructed from F plasmids
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YAC
DNA molecule that has a yeast ORI, pair of telomeres, and a centromere
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Ti plasmid
Plasmid that can be used to introduce DNA into plants
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Expression vector
Vector that allows the production of protein (i.e. transcription and translation)
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PCR Steps
- 1) Heat to 90-100 C for denaturation
- 2) Cool to 30-65 C for primers to anneal
- 3) Heat to 60-70 C for DNA synthesis
- Repeat
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PCR Limitations
- Requires knowledge of at least part of sequence for primers
- Taq polymerase is poor at proofreading
- Fragments larger than 50Kb cannot be isolated
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PCR as a diagnostic tool
Detects presence of a particular sequence, e.g. HIV
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Shotgun cloning
Clone all sequences in an organism into vectors
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DNA library
Collection of clones containing all the DNA fragments from one source
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Genomic library
Set of bacterial colonies or phages containing fragments in a DNA library
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cDNA library: isolating mRNA
isolation of mRNA using oligo(dT) chains
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cDNA library: making cDNA from mRNA
- oligo(dT) act as primers
- reverse transcriptase for DNA strand
- Rnase digests most of RNA strand
- Remaining RNA act as primers for second DNA strand
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In situ hybridization
A type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue
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Restriction Fragment Length Polymorphisms (RFLPs)
Variations in the patterns of fragments produced when DNA is cut with a restriction enzyme typically caused by mutation
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Dideoxyribonucleoside triphosphate(ddNTP)
No OH groups
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Sanger Method of DNA sequencing
Uses ddNTP to terminate synthesis of strands of different length which can be read by electophoresis to sequence
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Pyrosequencing
allows sequencing of entire genomes in a couple months
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DNA fingerprinting
Indentification of individuals by short tandem repeats (STRs)
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