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Gene Characterization


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DNA Sequencing
-paved way for deciphering human genome
-origionally done w/ maxam and gilbert's chem method
-now use sanger's dideoxy-sequencing method-simple, reliable, adaptable to high-throughput and automated approaches
synthesis of complementary strand
-Dideoxy relies on synth a DNA strand that is complementary to template(gene of interest)
-synth done w/ DNA polymerases
-primer-synthetic, single stranded oligonucleotides that are comp. to known sequence
-known sequence in flanking(before G of I) plasmid DNA or in region that has alread been sequenced
Place Primers based on- size of template DNA (1 primer=3-500bps), wether the seq of temp is already known
-Opposite template can be primed and synth in diff reaction w/ diff primers
random termination of synthesis
-*build 5' to 3'(add onto 3' end)
-neccessary for sanger''s method
-use dideoxy forms of nucleotide precursors (ddNTPs)-lack 3' OH (have H instead) -can't be added on to=termination
SEQUENCING-4 rxn's (ddATP,ddGTP,ddCTP,ddTTP)
-ratio of ddNTP to dNTP is low
-synth of comp strand is terminated at every spot randomly where there is an A in ddATP rxn, etc.
-rxn's give collection of termination products corresponding to every position in seq
High resolution sequencing gel
-use polyacrylamide gel-chem polymer formed by controlled crosslinking, separates small DNA's (<500bp)
-DNAs run toward + end, smaller moves farther
-place gel against x-ray film (orig primers were radioactive=extension prod's synth by DNA pol are too
-collumns for each ddNTP rxn (A,T,G,C)
-read up gel (read in 5' to 3' dirrection-b/c get longer as added to 3' end
-lowest=smallest=closest to primer
-as you get to bands going up gel=an A,T,G or C going from 5 to 3
-this gives 5' to 3' reading of comp strand, flip to get 3' to 5' reading of template, invert to get conventional 5' to 3' mode of template
automated DNA sequencing
-modified form of dideoxy sequencing
-ddNTP's that are tagged w/ diff florescent dyes(diff excitation/emission freq)
-all in same tube
-polyactylamide gel past detectors that give sequence
1)if seq is known, if so, whether it corresponds to previously id'ed gene
2)search for possible open reading frames(i.e. prot coding sequ)
3)examine for related seq or genes in same or diff species
human genome sequencing
NIH:-"walking" strategy-find one DNA frag, put probe on end and id next overlapping frag
Southern, Norther, and Western blotting
-based on ability to irreversably bind macromolecules(DNA, RNA, prots) to appropriate membranes, while they are still accessable to specific probes
-allows highly specific and sensitive detection quantification
-coupled with gel electrophoresis
-blotting=transfering to membranes
Southern blotting procedure
1)resolve or seperate DNA frags by electrophoresis through agarose gel(seperates large DNA-500-20kbps)
2)visualize by ethidium staining
3)gel is soaked in alkali sol'n *-DNA must denature to ssDNA prior to transfer to mem.
4)gel put in high salt sol'n-favors binding to mem.
5)paper towels draw transfer sol'n up through gel then mem-binds DNA frags to mem same spacial relationship
6)expose to UV light to crosslink mem
7)bake at 80 C to furhter fix DNA
*now it's ready for hybridization
*-DNA can be complex mix of Restriction frags of genomic DNA or simple i.e. confirming that cloned plasmid contains desired inserted DNA
Probing Southern Blot
1)radioactive probe is generated=short piece of synth ssDNA-oligonucleotide, or long piece from cloned cDNA or gene
2)mem briefly soaked in hybridization buffer
3)add radioac. probe
4)wash away remaining probe
5)lay on x-ray film
southern blotting results
-length of DNA in nts determined by inclusion of known DNA in gel
-it's semiquantitative-amt. of hybrid. probe is proportional to amt of target DNA-can determine relative levels of target sequencs w/in starting DNA sample
southern blotting Applications
-used while manipulating and cloning DNA to verify: authenticity of specific DNA frags. (including those by PCR), inclusion of cloned vector DNAs
-in conjunction w/ RE digests used to: give info on size and organization(exon/intron struc.), detect presence of foreign genes (transgenic mouse)
-clinical-test for:gene or chrom rearrangements, amplification, deletions or insertions that may characterize certain diseases (HER2-breast cancer)
Northern Blotting
-procedure is simillar to southern
-detect RNA rather than DNA
-no need to denature RNA-already ss
Northern Blot applications
-determine size of mRNA-amt of prob hybrid. reflects amt of target seq in orig RNA
-compare relative levels of mRNA in diff normal and diseased tissues, cell culture models, etc.
-examine regulation of expression of specific genes in development or in response to stim
-detect diff mRNA splice variants-due to diff sizes
-detect altered gene transcripts from gene deletions or insertions.
northern w/ southern app.
-can be used in conj. to deduce mechanistic basis of changes in gene expression.
EX: noth shows that HER2/neu gene is overexpressed in breast tumors, south distinguish b/w overexpression due to increased transc. or increased HER2/neu gene copy number
Western Blotting
-detection of proteins
-use anitbody directed against prot of interest as probe
-use polyacrylamide gel (<500bps)
-add antybody directed against target prot
-add beads that bond to antibody
-cetrigugation pellets beads
-discard sol'n
-resuspend beads and boil in SDS sol'n to release target prot
-ready for electrophoresis
SDS denaturation
-prots don't seperate right in electroph. b/c diff tertiary structure and diff charges
-SDS denatures(unfolds) prots and coats them w/ uniform neg. charge.
-gives them simmilar charge to mass ratio
Western Blotting
-run vertically through polyacrylamide gel
-use e-field to pull to mem and bind it irreversably
-soak w/ antibody dericted at target prot
-visualize antibodies w/ anti-IgG antibod directed at previouse antib. that is chemiluminescent
Western blot Applications
-determine size of prot
-deter. expression and/or reg or prot
-detect covalent modifications of prot(phosphorylation)
-assay for the prot during purification efforts
-polymerase chain reaction
1)high degree of specificity
2)exquisite sensitivity-from ability to amplify target seq more than a million fold
3)wide applicability
-allows mult cycles of comp strand synthesis
-defined length product dominates
PCR cycle
1)DNA heated to 95C to denature it
2)sample cooled to 55C to anneal oligonucleotide primers
3)adjusted to 72C and add heat-stable DNA pol to synth comp strands
MILLION FOLD-will run for 32 cycles(limiting enzyme-too much template)
AUTOMATED-Thermal cyclers, heat-stable DNA pol from bacteria grown in hot springs
first 3 PCR cycles
1) DNA denatured and primer added to on 3' end of both strands, replication cont indefinately(till pol falls off)
2)"unit length" product appears, ends=two primers,one to initiate synth, and one from end of temp, they are base paired w/ variable length strands
3)"unit length duplex" -dominates product
PCR applications
1)amp of specific DNA sequences prior to cloning(obviating need for library screening)
2)amp of gene seq for hybridization probe (elim need fro cloning)
3)amp of seq fro direct sequencing(look for disease assoc. mutants
4)extremely sensitive test for viral genomes or transgenic constructs
5)detect specific mRNAs via amp of reverse transcriptase-gen cDNAs, and differential PCR
pcr limitations
1)amp is limited to few kbases-enzymes lack staying power to cover large frags
2)contamination is MAJOR problem(prob in HIV test,forensics)
3)assay is not quantitative
real time PCR
-add 3rd "reporter" oligonucleotide annealed to comp seq of DNA
-has fluorescent on 5' end and fluor. quencher on 3' end
-Taq pol gets to 5' end and knocks fluor. off of DNA->it fluoresces-quantify amt. of fluor
-make standard curve of known values to compare to
***GENOMIC PCR-determine # of gene copies(HER2)
***RT-PCR-quantitate viral load(HIV), OR compare expression of key genes in normal vs. disease

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