Powerpoint 2 Chapter 5 Flashcards
? cleave polypeptides at specific symmetry sites
restrictions enzymes
southern blots identify specific ? sequences
DNA
northern blots identify specific ? sequences
RNA
chemical synthesis of DNA
a deoxyribonucleoside with DMT and BCE attached couples to OH group of resin nucleotide. Elongation continues by deprotection with dichloroacetic acid
Sanger sequencing
primer is atached to DNA template to be sequenced and a DNA polymerase is added along with labelled sNTPs and a dideoxy dNTP.
PCR
add excess flanking primers of desired DNA sequence. heat to separate strands. cool to anneal primers. synthesize new DNA. heat to seperate. cool to repeat synthesis.
vector
a DNA molecule that can replicate autonomously
Plasmids
circles of DNA that act as accessory chromosomes in bacteria, can replicate independently.
plasmids cut by ? for recombination
restriction enzymes
cut plasmids can be annealed using ?
DNA ligase
what if DNA is not naturally flanked by appropriate restriction sites?
DNA linker containing restriction site added to ends of DNA
insertional inactivation
inserting DNA within a gene to inactivate
polylinker
contains many unique restriction sites
benefits of bacterial and yeast artificial chromosomes
allow cloning of larger inserts of DNA
requirements to clone small DNA fragment in a large DNA collection (2)
specific oligonucleotide probe for gene of interest and a DNA library that can be screened rapidly
how is a specific probe obtained?
by synthesizing a oligonucleotide sequence of codons complementary to part of the amino acid sequence of a protein
preferred amino acids of Probes
methionine and tryptophan because they only have one codon
lambda phage
a virus that is used as a vector for cloning in E. Coli
creation of genomic library
genomic dna is fragmented and joined to lambda phages. lambda phages infect E. Coli and replicated. The phages lyse the E. coli host. Sufficiently large number of virus particles to ensure entire genome is represented.
how to screen a genomic library for a specific gene?
plate phages of genomic library on lawn of bacteria. Apply nitrocellulose paper to make copy. Add NaOH and labelled probe. Add x-ray film to identify label.
oligonucleotide directed mutagenesis
a primer containing a mismatched nucleotide is used to produce a change in DNA sequence and in turn a single amino acid substitution
cassette mutagenesis
deletion of gene in a plasmid by cutting with restriction enzymes, purifying the remaining fragment, and adding a new gene (cassette) through ligation to reform the plasmid
recombinant HGH replaced HGH isolated from ?
cadavers
transcriptome
the entire set of RNA molecules in a population of cells
size of genome does/does not reflect number of genes
does not
SINES
repetitive DNA elements
number of pairs of autosomes in human genome
22
changes in gene expression in response to stimuli can be monitored on ?
microarrays/ gene chips
microarray
oligonucleotides affixed to suport, labelled cDNA allowed to hybridize to reaveal expression level for each gene, identifiable by known position on microarray
episomal
not integrated into bacterial genome
rat growth hormone containing DNA can be injected into fertilized mouse egg with ? producing transgenic mouse that produces 500 times mores somatotropin
micropipette
Ti plasmids
used to introduce foreign genes into plants
types of site-specific double strand breaks
non-homologous end-joining repair, homologous recombination repair (better)
non-homologous end-joining repair
error-prone. used for gene disruption or knockout
homologous recombination repair
error-free. used for gene replacement (or knockout)
RNA intererence
introduce double stranded RNA. cleaved by DICER into 21bp siRNA. siRNA loaded into RISC protein. one strand cleaved and removed. remaining strand recognizes and binds mRNA which it in turn cleaves and releases.
(gain/loss) of myostatin leads to bulked up mice
loss
? factors control gene expression
transcription
engineered transcription factors implementing domains such as ? or ? can be used to activate or repress gene expression
zinc finger or tal effectors
Zinc finger nuclease
generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain
CNBr cleavage
carboxyl side of Tryptophan
Trypsin cleavage
carboxyl side of lysine and arginine
chymotrypsin cleavage
carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine
carboxypeptidase A cleavage
amino side of C-term Amino acid (excluding arg, lys, pro)
