Next Generation Sequencing, Gene Mapping, and Epigenetics Flashcards
three things for the intial discovery of a DNA alteration
- well defined phenotype so you get a sense of what to look for
- family based genetic analysis
- possibly comprehensive analysis of the genome
array based analysis
- hybridization assay
- patient sample is characterized by its complimentarity with a known sequence
sequencing
- broad look of how it is done
- gold standard
- done by monitoring the incorporation of each nucleotide during in vitro DNA rep
- sanger is the gold standard
comparative genomic hybridization DNA arrays
-identify differences between patient and a control
detects deletions and duplications too small to be evident on cytogenic methods
-change could be a polymorphism or the reason the test is being done
-make your DNA spots in seqquential order going down the chromosome
Single Nucleotide Polymorphism DNA analysis
- what is the principal
- population differences
- what are SNP’s
- use
takes advantage of neutral single nucleotide differences across the genome
- these are different for each population
- SNP’s are single nucleotide differences that are present in at least 1% of the reference population
- can be used to map copy number changes and loss of sequence
limitation of CGH and SNP arrays
-can not provide information on chromosome rearrangement that do not produce gains or losses of the DNA sequence
SNP analys can be done using two different types of technique
-arrays or sequencing
applications of SNP genomic mapping
- analysis of suspected genomic lesions in one patient
- genome wide association studies
- ancestry genotyping using ancestry informative markers
next generation sequencing or massively parallel sequencing
- multiple subsequent sequencing reactions in an automated form
- high throughput may be achieved with some loss of accuracy
- immense data analysis
- several techs available
preparing DNa for NGS
-must be fragmented and usually modified with handles to allow for manipulation
illumina NGS sequence by synthesis
- fragment the smaple
- attach handles (adapters)
- capture the fragments on a solid surface
- amplify the fragments
- add labeled chain termination sequence
- wash, take pic and repeat
bringing down the time and cost of NGS
- DNA fragments are pre-enriched for coding sequences as to not have to sequence the introns
- this is called whole exome sequencing
epigenetics
heritable changes in gene expression that arise from changes in the chromosome without alteration of the DNA sequence
-DNA methylation
-Three independent DNa methyltransferases
- DNMT3A and B methylate DNA de novo (neither strand is methylated to begin with)
- DNMT1 methylates hemimethylates DNA after replication
where methylation is found
-characteristics of methylation
- occurs at CpG sites outside of CpG isalnds (found on promoters)
- most CpG sites are methylated
- methylation typically corresponds to long term epigenetic memory.
- once a site is methylated, it will remain that way for manny cel cycles
pathogenic effects of methylation
- methylated genes can act just like a loss of functions mutation
- for instance if a tumor supressor were to become methylated
how do we test for methylation
we can use bisulfite treatment to turn unmethylated C’s into U’s leaving the methylated C’s the same so they can be spotted
