Molecular Biology Tools Flashcards
Expression of Recombinant human insulin in E. Coli
1) insulin mRNA is copied by RT into cDNA
2) end of insulin cDNA is digested
3) insulin cDNA is ligated into bacterial vector that has also been digested to make the recombinant DNA.
4) bacterial cells get transformed
5) Bacterial cells produce insulin
Reverse Transcriptase
RT binds
2) mRNA is annealed to synthetic primer
3) synthesis of cDNA in 5’ to 3’ direction
4) makes dDNA-mRNA hybrid
5) mRNA is digested with alkali
6) primer is ligated to 3’ end of cDNA
7) DNA pol extends from primer to yield ds DNA
Restriction Enzymes
recognize and cleave recognition sequences in DNA
found in bacteria
makes both sticky ends and blunt ends
Recognition sequences
usually 4-6 nts and are palindromic
DNA ligase
joins 5’ phosphate with 3’ OH to form phosphodiester bond
can join fragments from two different DNA
Plasmid
extra chromosomal DNA in bacteria that replicates independently, does not integrate into the chromosome
used in vectors
confers antibiotic resistance
contains: OR, restriction sites, selectable marker
simple to clone, but low transformation efficience
Antibiotic resistance
plasmid vector contains antibiotic resistance gene making the transformed cell resistant. plated on agar medium containing antibiotic for selection
Bacteriophage
infect E coli with high efficiency
introduction of recombinant vector
kills host
25kb insert
Cosmids
hybrid of plasmid and bacteriophage
does not kill host
uses plasmid OR
carry up to 45kb DNA
CRISPR
confers resistance to foreign plasmids and phages
cut exogenous genetic elements similar to RNAi
regulates Cas9 delivery
Cas9
is an RNA guided DNA endonuclease
unwinds foreign DNA or plasmid and checks to see whether it is complimentary to 20bp spacer region of guide RNA.
if complimentary, Cas9 cuts it to cleave it.
Restriction Digestion
1) bacteria containing recombinant plasmids are grown as clones
2) DNA is isolated from multiple clones and digested with enzymes
Pattern of restriction enzymes shows if DNA segment of interest has been inserted
cathose is ______
negative
anode is
positive
Intercalating dye
insertion of dye between molecules of the planar bases of DNA Ethidium bromide (exposed to UV)
Hybidization
cones are blot transfered to membrane and DNA is denatured and fixed onto surface.
add radioactive probe and allow to hybridize (temp just below Tm)
exposed to X ray and see where the hybridization occurd.
Tm =
2°(A+T) + 4°(C+G)
Southern Blotting
DNA fragment separation
labeled with radioactive DNA probe
Northern Blotting
RNA fragment separation
SDS-Page
separates proteins based on size and forms a negatively charged complex with proteins to allow them to migrate through a polyacrylamide gel.
Probes used in Blots
cDNA for Genes for Southern and Northern
Antibody for Western
1) specific length 2) specific sequence 3) radioactivity for detection 4) must be added in sufficient quantity to compete with other binding
Microarrays
developed to simultaneously analyze expression of thousands of genes at once.
1) isolate total mRNA in each condition
2) reverse transcribe cNDA labeled with fluorescent dye
3) mix
4) hybridize to DNA in microarry
5) wash
6) measure fluoresce one each spot.
traditional DNA seqeuncing
2’3” dideoxyribose which does not have 3’ OH, so it cann’t be extended.
1) combine unkown DNA with pol, primase, dNTS, in four containers each with a specific A, T, C, G, of ddNTP
2)DNA syntehsis occurs
3) separate fragment by electrophoresis
sequence is the compliment of the unknown strand
PCR
1) DNA denatured at 95° and cooled to 55° to allow primers to hybridize
2) Temp is raised to 72° and polymerase makes copy to 3’ end
3) repeat cycle
gives 2^N copies
taq
heat safe DNA polymerase used in PCR
Restriction Fragment Length Polymorphisms
1) disease alleles cause either a gain or loss of restriction endonuclease cleavage
2) for screening of HbS mutation in sickle cell anmeia, destoys restriction site for MstII.
3) digest patient DNA with diagnostic RE followed by southern or PCR analysis to detect altered size.
mutated MstII restriction site
in Sickle Cell anemia
Changed from A to G
produces two 1.3 kb segments
normal has 1.1 and .2 segment
Allele Specific PCR
used to detect small mutations (point mutations, insertion or deletions)
primers are designed to hybridize to mutant but not normal allele
can test for multiple mutations at the same time by designing so that each PCR product is a different size.
Cystic Fibrosis
CFTR gene the fourth Amino acid is normally TTT or Phe.
in delta F508, common mutation in cystic fibrosis, mutations gives rise to GGT and Glycine. must change the mutant primer.
DNA genotyping
use microarray to determine the status of a single NT polymorphism at specific base location
determines mutant status (heterozygosity) as well as halotype status
1) add anneal specific probe to genomic DNA
2) fill gap and ligate and do PCR to create fluorescent bar coded probe for each allele
3) measure fluorescence
Paternity Testing
DNA fingerprinting by variable number of tandem repeats.
PCR or restriction digestion followed by south blotting of region with repeats
Short Read sequences
DNA sequencers that produce lots of short 100 bps reads in single run.
low error rate
used to provide high coverage over specific set of DNA template from complex mixture.
Illumina
Long Read Sequences
can read 10,000Bp in length
high error rate
useful in human genetics for linking contiguous polymorphisms, measuring diveristy of mRNA isoforms,
watches single DNA pol incorporate fluorescent NTs.
Read depth
Coverage
number of times a base in the genome was independently sequenced. high read depth improves error rate
Error Rate
measurement of quality of converting signal into accurate prediction of NT.
Continguity
see the recombination events between male and female gametes. Recombination cross over tend to be long distances apart.
short read are too short to link variants in the same haplotype block
long reads can sometimes link variants together
Fragmentation of DNA
is pseudo-random, meaning ends vary
Single nucleotide polymophisms
are detected through reading multiple fragmented DNA and seeing how often the mutation occurs.
what if 50% of fragmented DNA has mutation
either high error rate or heterozygous mutation
Exome Sequencing
used to isolate DNA fragments that contain protein coding sequences for sequencing
useful in sequencing mendelian where variants are rare and not likely to exist in database
1) use oligonucleotide probes that are complimentary to regions of interests that hybridize to DNA
2) fish out DNA fragments to be sequenced
Variants of unknown significance
exome sequencing has produced large catalog of variants within protein coding regions, but it is difficult to predict with high confidence the functional impact.
how do we interpret variants of unknown sigificance?
1) genetic analysis and looking at each variant (valid, but low throughput)
2) computation prediction - do a mutation and see if it does something to structure - not valid, but high throughput
3) massive parallel functional analysis - high validity and high throughput
Parallel Functional Analysis
make a tube with all possible variants that have single mutation
measure how many variatns are in tube before and after. if this changes, it indicates gain or loss of function of variant.
Genomic DNA footprintingq
treat cell with low concentration of DNAse I that cuts based on accessibility, DNA fragments are collected and sequenced to identify regulatory sites.
