Bio115 29-31

chemicals with structures similar to any of the four standard bases

base analog for thymine which can mispair with guanine leading to a transition mutation

base analog of adenine which can mispair with cytosine -> transition mutation

guanine->O6-Ethylguanine: pairs with thymine -> transition mutation

cytosine->Uracil: pairs with adenine -> transition mutation

cytosine->Hydroxylaminocytosine: pairs with adenine -> transition mutation

reactive forms of oxygen that damage DNA and induce mutations via chemical change

molecules which insert into DNA in place of nitrogenous bases causing insertions and deletions, e.g. proflavin and acridine orange

1927: showed mutations in fruit flies inducible by radiation

alter base structure, break phosphodiester bonds, and even cause double-strand breaks

less energy, but still mutagenic

can be caused by UV light; create covalent bonds between bases which block replication

eukaryotic system of eta polymerase that can bypass pyrimidine dimers

Replication errors, including mispaired bases and strand slippage

Pyrimidine dimers; other specific types of alterations

Abnormal bases, modified bases, and pyrimidine dimers

DNA damage that distors the double helix, including abnormal bases, modified bases, and pyrimidine dimers

• 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

removes nucleotides usually at end of DNA strand

replaces nucleotides

seals nick in sugar-phosphate backbone

• 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

an enzyme that uses light energy to break covalent bonds in pyrimidine dimers

• 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

removes nucleotide usually in middle of DNA strand

• 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

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.

Two DNA strands are connected thru covalent bonds. Not much know about this.

• Detection
• Excision
• Polymerization
• Ligation

Damaged section of DNA is recognized

DNA repair endonucleases nick phosphodiester backbone on one or both sides of the DNA damage and one or more nucleotides are removed

DNA polymerase addes nucleotides to the newly exposed 3'-OH group by using the other strand as a template and replacing the damaged nucleotides

DNA ligase seals the nices in the sugar-phoshate backbone

How detection and excision are accomplished

autosomal recessive condition caused by nonfunctional repair mechanism for pyrimidine dimers

Created first recombinant DNA molecule

Set of molecular techniques for locating, isolating, altering, and studying DNA segments

• 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

• Enzymes that recognize and make double-strand cuts in DNA at specific nucleotide sequences
• Produced naturally by bacteria to defend against viruses

Cut outside recognition sequence

• Cuts within recognition sequence
• Used in molecular genetic work
• Names indicate original bacteria
• More than 800 isolated

• Palindromic recognition sequence
• Fragment end either cohesive or blunt

Staggered cut -> sticky ends

Reaction of mixture of DNA, buffer, restriction enzyme, water heated at about 37 C

Standard technique for separating molecules on basis of size/electrical charge

polysaccharide isolated from seaweed

DNA fragments move to positive pole with smaller fragments moving faster

fluorescent or radioactive DNA or RNA fragment complementary to sequence of interest

Transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by radioactive probe

• 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

• Transfer of proteins from gel to a membrane
• Probe is usually an antibody
• Determine size of protein
• Pattern of protein's expression

create identical copies of a piece of DNA

DNA molecule into which a foreign DNA fragment can be inserted for introduction into and replication in a cell

• Origin of replication
• selectable marker
• one or more unique restriction sites where DNA can be inserted

• Plasmid, e.g. pUC19
• Bacteriophage
• Cosmid
• BAC
• YAC
• Retroviral vectors
• Transposons
• Expression

The capacity of bacterial cells to take up DNA from the environment

Plasmids that are packaged into empty viral protein coats and transferred to bacteria by viral infection

Originally contructed from F plasmids

DNA molecule that has a yeast ORI, pair of telomeres, and a centromere

Plasmid that can be used to introduce DNA into plants

Vector that allows the production of protein (i.e. transcription and translation)

• 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

• Requires knowledge of at least part of sequence for primers
• Taq polymerase is poor at proofreading
• Fragments larger than 50Kb cannot be isolated

Detects presence of a particular sequence, e.g. HIV

Clone all sequences in an organism into vectors

Collection of clones containing all the DNA fragments from one source

Set of bacterial colonies or phages containing fragments in a DNA library

isolation of mRNA using oligo(dT) chains

• 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

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

Variations in the patterns of fragments produced when DNA is cut with a restriction enzyme typically caused by mutation

No OH groups

Uses ddNTP to terminate synthesis of strands of different length which can be read by electophoresis to sequence

allows sequencing of entire genomes in a couple months

Indentification of individuals by short tandem repeats (STRs)