Next Generation Sequencing, Gene Mapping, and Epigenetics

Question 1

three things for the intial discovery of a DNA alteration

Answer 1

• well defined phenotype so you get a sense of what to look for
• family based genetic analysis
• possibly comprehensive analysis of the genome

Question 2

array based analysis

Answer 2

• hybridization assay

- patient sample is characterized by its complimentarity with a known sequence

Question 3

sequencing

• broad look of how it is done
• gold standard

Answer 3

• done by monitoring the incorporation of each nucleotide during in vitro DNA rep
• sanger is the gold standard

Question 4

comparative genomic hybridization DNA arrays

Answer 4

-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

Question 5

Single Nucleotide Polymorphism DNA analysis

• what is the principal
• population differences
• what are SNP’s
• use

Answer 5

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

Question 6

limitation of CGH and SNP arrays

Answer 6

-can not provide information on chromosome rearrangement that do not produce gains or losses of the DNA sequence

Question 7

SNP analys can be done using two different types of technique

Answer 7

-arrays or sequencing

Question 8

applications of SNP genomic mapping

Answer 8

• analysis of suspected genomic lesions in one patient
• genome wide association studies
• ancestry genotyping using ancestry informative markers

Question 9

next generation sequencing or massively parallel sequencing

Answer 9

• multiple subsequent sequencing reactions in an automated form
• high throughput may be achieved with some loss of accuracy
• immense data analysis
• several techs available

Question 10

preparing DNa for NGS

Answer 10

-must be fragmented and usually modified with handles to allow for manipulation

Question 11

illumina NGS sequence by synthesis

Answer 11

• 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

Question 12

bringing down the time and cost of NGS

Answer 12

• DNA fragments are pre-enriched for coding sequences as to not have to sequence the introns
• this is called whole exome sequencing

Question 13

epigenetics

Answer 13

heritable changes in gene expression that arise from changes in the chromosome without alteration of the DNA sequence
-DNA methylation

Question 14

-Three independent DNa methyltransferases

Answer 14

• DNMT3A and B methylate DNA de novo (neither strand is methylated to begin with)
• DNMT1 methylates hemimethylates DNA after replication

Question 15

where methylation is found

-characteristics of methylation

Answer 15

• 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

Question 16

pathogenic effects of methylation

Answer 16

• methylated genes can act just like a loss of functions mutation
• for instance if a tumor supressor were to become methylated

Question 17

how do we test for methylation

Answer 17

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