Methods Exam 1

shotgun sequencing

gDNA cut into many small fragments → finds overlaps to order the clones and assemble long sequences by computer, faster and cheaper

map-based sequencing

combines generated genetic and physical maps with newly generated sequences, cut with restriction sites → overlaps used to form contigs, time consuming and expensive

Sanger sequencing

creates new strand from primer and template to sequence, can only generate single sequence at a time, reads 800-1000 nucleotides from template, labor intensive and difficult to read early gels, error rate 1% (worse at end) → need for more capacity, better costs, and speed

DNA fragmentation, in vivo cloning and amplification in E.coli, chain termination with ddNTPs (cleavable fluorescent ddNTP with irreversible chain terminator H), electrophoresis (1 read/capillary), 1 fragment template

can sequence up to 1000 bases, shotgun sequencing breaks up larger sequences into random fragments, requires amplification or plasmid cloning, used when you need single gene region for a few samples, barcoding (simple identification), characterizing clones

Next Gen sequencing

involves DNA fragmentation, in vitro adaptor ligation with different adapter at each end, amplify fragments in clonal clusters using PCR (polonies), cyclic array sequencing with reversible chain terminators, many reads, sequencing by synthesis using rtNTPs with many DNA fragment templates

can sequence 20-250bp total output 150 trillion bp, very accurate and inexpensive, not great for repetitive genomes or de novo assembly, used when you need lots of gene sequences, short read map to exons, struggle to resolve differentially spliced mRNAs

pyrosequencing

adds one nucleotide at a time and looks for emission of light from release of pyrophosphate where each nucleotide gives different signal

proton detection (Ion Torrent)

detects H ions released during DNA synthesis where each nucleotide gives different signal, replaced by reversible dye terminator (fluoresecently labeled nucleotides)

illumina sequencing

fragment DNA and add dsDNA adapters to end to prep library, indexes are barcodes that allow mixing of libraries in single sequencing run (multiplexing), each sequence amplified in situ to create PCR colony (ds bridge amplification for cluster generation), each nucleotide fluoresces different color

3’-O-blocked reversible chain terminators

3’-OH replaced with OR group so fluorophore and protecting group can be removed after each nucleotide read

3’-unblocked reversible terminator is where dye is terminator and is cleavable, 3’-OH intact but not initially accessible

Third Gen sequencing

long sequencing, sequences molecule directly (no amplification) so very fast

can sequence up to 1 million bp, expensive and lower accuracy rate, good for repetitive and novel genomes, used with long sequencing reads so easier genome assembly and confident isoform sequencing, longer reads easier and faster to assemble and can span long repetitive regions

Oxford Nanopore sequencing

DNA passes through a pore pulled through by an electrical potential, each nucleotide affects current across membrane differently and read as A G C or T, require minimal prep, no amplification (single molecule), integrated directly with computer

1.) isolate intact RNA for qRT-PCR

lyse cells in Buffer LBP before to inactivate RNases → pass through gDNA removal column (prevent false positives in qRT-PCR) → RNA spin column to remove other contaminants → elute isolated RNA (>200 nucleotides includes mRNA) with water

2.) quantitate RNA using Nanodrop

determines RNA concentration and purity with A260/A280 ratio of 2

3.) assess RNA quality

using gel - top and bottom bands are 28S and 18S rRNA, clear bands with no smearing show intact RNA can assume mRNA intact too since rRNA is 90% of RNA

using Bioanalyzer nanochip - 2 strong peaks for 28S and 18S rRNAs, RIN=10 shows intact RNA

Northern Blotting

1-10 genes analyzed, RNA hybridized with radiolabeled cDNA probe, detects size of transcript and shows variants (gene family, alternative splicing), tedious, not sensitive or quantitative, replaced by qRT-PCR

qRT-PCR

analyze hundreds, fast and sensitive, allows comparison of multiple samples (tissue samples, biopsies), use in this lab to analyze gene expression using mRNA

Microarrays

convert RNA to cDNA → tag with fluorescent dyes → hybridize to microarray with known probes, tedious, affordable, less sensitive, comparison of all genes between 2 samples

RNA-seq

convert RNA to cDNA library → sequence and align to reference genome, sensitive, identifies novel transcripts and alternative splicing, more detailed, multiple samples compared

noncoding regulatory RNAs

regulate gene expression by silencing genes - RNA interference (RNAi), 18-25 nucleotides, target degradation, act through RISC and processed by DICER

siRNA

defends genome from nucleic acids like viruses/transposons, from long dsRNA, not in mammals, fully complementary, 1 target

miRNA

transcribed from genome that regulates gene expression, from longer ssRNA that folds into hairpins, regulate endogenous genes (from own genome), found in mammals, translation inhibition, partially complementary, many targets

DICER

processes dsRNA (siRNA and pre-miRNA) to be correct length for RNAi

RISC

RNA-induced silencing complex, uses siRNA or miRNA to guide it to mRNA, then degrades target mRNA

RNAi

siRNA and miRNAs affect mRNA translation/stability, results in loss of protein by mRNA degradation or translational blockage → gene silencing

can be used to downregulate gene (knockdown), alternative to using cell lines from knock-out animals

primer design

designing priers to check for gene editing efficiency by PCR amplifying edited region (CRISPR edit efficiency), gRNA designed to edit unique PstI site in sequence (target), if PstI site present (not edited) then PstI digestion will produce 2 bands on gel

PCR primer parameters

• unique oligonucleotide sequence - 100% query cover and identity means all nucleotides of primer match sequence on BLAST, low E value (# of hits expected by chance when searching database of particular size), bovine HSP70 gene not same as murine so primers might not bind

• 17-30bp long

• no self complementarity to prevent hairpins and self dimers - hairpin ∆G should be large positive so hairpins unstable and don’t form

• no complementary between 2 primers to prevent primer dimers

• ∆G low negative like -3kcal/mol - too negative means more secondary structures which are undesirable for PCR

• avoid single base runs and dinucleotide repeats

• GC content about 50% - more stable more 3 H-bonds

• Tm (temp where 50% of DNA duplex dissociated) - 52-58°C, annealing temp 5-7°C below primer, primers have similar Tm

specificity of PCR depends on primers, make sure primer will amplify region with PstI site, make sure PstI site not within primer

primer binding

reverse primer complementary to reverse of sense strand and binds to sense sense, forward primer is same sequence as sense strand and binds to antisense strand

sequencing to look for edits

plasmids sequences using T7 promoter primer, 5’→3’ ATG is sense strand +/+ (start of gene), GCA is antisense +/- (end of gene), PCR product can insert in 2 different orientations but data always 5’→3’

.ab1 files used to observe chromatograms - good sequence has evenly distributed peaks, each peak single color, no noise or background

in BLAST, look for mutations around PstI site, further mutations are probably not from CRISPR