ch 13 plus whateveah Flashcards
biochemgel electrophoresis
analyze, Identify, purify
separates based on size (long at top), charge, or conformation
DNA, RNA, proteins
agarose gel electrophoresis used for
nucleic acids- bad resolution- horizontal gel
pulsed field gel electrophoresis
pulses electric charge, for larger fragments of DNA
polyacrylamide gel electrophoresis
more precises
nucleic acid and proteins
vertical gel
how is DNA sorted in electrophoresis
big thing at top (near well and negative charge) smaller thing at bottom
ethidium bromide
glows and shit ehehehehhee
can purify DNA after sorting
southern blotting and probe
detect DNA, ran on gel electrophoresis and transfer to membrane
Probe is single stranded complementary DNA or
RNA fragment
northern blotting
detect RNA, like southern
Probe is single stranded complementary DNA or
RNA fragment
western blotting
detect protein, need to denature ts first, Primary antibody to specific polypeptide,
Secondary antibody to detect/amplify primary, acrylamide
probe
nucleic acid that has same or similar sequence to gene of interest, labeled and shit,
hybridize with the target
antibody
detect antigen
antibody composition
2 heavy chains, 2 light chains, antigen binding site
polyclonal antibody is obtained from
injecting an animal with the antigen and bleeding it
secondary antibodies
target fc fragment (constant region)
PCR
amplifies specific sequence of DNA
needed for PCR
DNA poly, DNA template, DNA primer, dNTP, buffer/cofacters
main polymerase used for PCR
Taq DNA poly for thermus aquaticus
PCR steps
Cycle: Denature, Anneal, and Elongation
PCR can be used to identify
trinucleotide repeats
PCR uses
genotyping, mutation detection, forensics, paternity testing, ancestry, trace disease, sequencing, cloning
RT-PCR
evil RNA PCR
RT-PCR great for
single/few gene, check transcription levels, test for RNA viruses
manual sequencesing/ Sanger is how logn and requires what gel
Dna syntehsis reaction, short (500bp), need polyacrlyamide gel
Sanger sequencing needs
DNA poly
DNA template
free 3’ OH
dNTPs
ddNTPs
primer
Sanger steps
elongation, random termination, sorting via gel elctrophoresis, shows complement
why is ddNTP a replication terminator
no have 3’ OH
next gen sequencing
sequining of spatially seperated, clonal amplified DNA templates in array at same time,
for whole genomes
automated DNA sequencing
ddNTP is tagged with diff colors, loaded in capillary array to sort, laser beams, electro-gram converted to sequence using computer
illumina sequencing
- amply- bridge PCR
- sequences- fluorescence signal made by reversible terminators
- analyze
RNA sequencing is for
gene expression, changes in whole genome expression
recombinat DNA tech and products
combines DNA form different sources, makes vectors/plasmids
molecular cloning is selective - of a
selective amplification of a particular gene or DNA segment
transgenic organisms
genome altered with frogmen DNA sequences by artificial means- shows where genes are expressed and expresses recombinant protiens
why clone a gene?
amply DNA
DNa sequencing
create gene alteration
study mutations
1st step in making transgenic organisms
(5)steps to molecular cloning
- obtain DNA segment
- select small molecule of DNA that can self replicate
- join DNA
- put into something
- Id host cells that have this DNA
enzymes needed for recombinant DNA
ligase, restriction endonuclease
DNA ligase
joins two pieces of DNA by forming phosphodiester bonds
Restriction endonuclease
recognized a sequence and cuts at a recognition site
restriction endonuclease original purpose
restrict/ prevent viral infection by degrading invading nucleic acid
methylase activity
addition of methyl group to protect these sites in DNA sensitive to attack by restriction endonuclease
methylate typical target
adenine methylation to 6 methyl adenine
most common restriction endonuclease
type II, we use ts a lot
type II endocnulcease
Recognize and cut at recognition site, makes sticky/blunt (straight) ends
how to get sequences for amp/manipulate
nonspecific shearing from genomic DNA or cDNA