W1L3 genomic technique Flashcards
What is the goal of genetic technique
-Probing genetic at distinct level and identify genomic loci w/ DNA change that contribute to trait:
-carry out by Contrasting effect of variants in distinct regions of DNA on traits:
* Variants that affect protein structure / function within organisms
* Variants that affect protein expression within organisms
* Different levels of environmental influences
Underlying genetic contributor of a phenotype
-Simple traits link DNA variant through protein structure, sequences and fuction, most are in coding sequence with phenotype (but only 2%)
-However most trait including disease are complex with genetic variation in non-coding loci
How can arrangement of DNA affect expression
Densely packed heterochromatin - suppressed gene expression
Euchromatin - lightly packed, actively expressing
Gene accessibility depend on DNA structure
-Some other factor: DNA looping, interchromosomal contacts, Epigenetic modification
Past and current gene technique
-Nothern blot (Alwine 1977)
-Reverse transcription of mRNA ( Weis 1992)
-Qualitative PCR of DNA (Higuchi 1993)
0measurement at single cell, single molecular resolution
-improved affordability of techniques increase utility and sample size
Forward genetic
- start with a traits and then identify underlying genetic contributor
- identify genes that might contribute to the trait
unbiased mutagenesis screen method
Chemical random mutagenesis targeting coding sequences
1. Single nucleotide mutations
2. Screen for phenotype
3. Deep sequencing + complementation
-Random insertional mutagenesis:
1. Insert DNA (e.g. GFP reporter) - usually null phenotype
2. Screen for phenotype
3. Identify site of insertion to ID gene. If a gene is important, then it will be knock out, measurable
Example for unbiased chemical mutagenesis screen
- Chemical random mutagenesis of male. Every cell will have different mutations!
- F1 - Offspring of parental outcross arise from 1 sperm and every cell will have the same mutations.
- F2 - Offspring of F1 outcross will have half of the offspring WT and half heterozygous for every mutations. These are the F2 families.
- F3 - Incross of the F2 families will result in Mendelian ratios, for recessive mutations - 1/4 of crosses have two heterozygous parents and 1/4 quarter of the offspring are homozygous: SCREENING!
What is compllimentaion
-asssay between novel mutant to a known mutants relevant to the phenotype
-If mutations are in different genes (A & B), the wildtype allele is supplied by the other parent in the heterozygous offspring.> MUTATIONS COMPLEMENT
-If mutation are in the same gene, no WT mutant> mutation do not compliment
Identifying genetic variants contributing to complex traits
- Sequence and compare DNA sequences (BLAST)
- each persons have 3-5 million nucleotide variation
-GWAS to identify underlying genetic differences between groups of people with a distinct trait differences. Use manhattan plot to analyze, peak mean linkage between gene and trait. This does not mean causation
Reverse genetic
-start with a gene of interest and understand how it contribute to a phenotype
-Reverse genetics allows us to study the cellular, organ and behavioural phenotype to assess the function of one particular gene.
Step up reverse genetic
How does your gene candidate or SNP contribute to the trait? (Strong or weak)
Monogenic / simple functional gene studies are convenient for reverse genetic
Transcriptional regulation - transcriptomics: changes in gene expression can involve a whole downstream network of expression changes
Look at: RNA sequencing, Expression profiling, Transcriptional regulation
RNAseq workflow
-RNA is converted into a cDNA fragment library.
-Sequencing adaptors are added and the each fragment is sequenced.
-Reads are aligned to a reference genome.
RNAseq Concepts
Sequencing depth (total number of reads mapped to genome)
Gene length (exonic length vs fragment length)
Gene counts (product of depth and gene length and splice variants)
Method for RNAseq analysis for biological meaning
Differentially expressed gene:
-heat maps
-volcano plots
Expression profiling: gene ontology
Measuring the accessibility of DNA
Assay for Transposase-Accessible Chromatin
-Tn5 transposase homodimer can attach to accessible DNA and tag it
-After fragmentation tagged DNA is amplified and sequenced
