genetics topic 13

DNA microarray purpose

determine the genotype at an SNP

Genomic resequencing purpose

to identify SNPs and CNVs

RNA microarray purpose

To measure level of gene expression

RNA sequencing purpose

to measure amount of transcription of genes expressed

Does DNA microarray look at:

level of gene expression

alternate spicing

CNVs

SNPs

SNPS

Does RNA microarray look at:

level of gene expression

alternate spicing

CNVs

SNPs

level of gene expression

Does genomic resequencing look at:

level of gene expression

alternate spicing

CNVs

SNPs

CNVs and SNPs

Does RNA sequencing look at:

level of gene expression

alternate spicing

CNVs

SNPs

level of gene expression, alternative splicing, and SNPs

How does DNA microarray work

1. have DNA chip with small synthetic ssDNA sequences that are complimentary to genomic sequence with a SNP

2. DNA of interest is fragmented and separated into single strands then hybridized to the single stranded synthetic DNA on the slide

3. the empty position due to the DNP is revealed when the slide is washed and fluorescently labeled nucleotides are added

4. each color reflects the genotype of the individual at SNP. if homo- color will match one of the four, if hetero- spot will be a mix of two colors

How does Genomic Resequencing work

1. Use illumina sequencing

2. sequence is compared to a reference genome and aligned based on similarities. If homo- all reads will be the same, if hetero- reads will be 50% one nucleotide and 50% another

How does RNA microarray work

1. have a synthetic single stranded DNA bound to a slide

2. extract RNA from the sample of interest, it is reverse transcribed to make cDNA, add fluorescent label, then hybridize to the slide

3. highly expressed genes have RNA bound and are a bright color

can be used for competitive hybridization which shows relative expression of each gene between two samples. in this case you use two colors like red and green, a combo of the two is yellow, no expression is black

How does RNA sequencing work

1. collect mRNA from a tissue and reverse-transcribe it to make cDNA

2. sequence using illumina which results in reads which are then aligned to the species’ reference genome sequence

3. use read depth for each gene to assess level of expression and SNP if found in exons of expressed genes