BIOL200 gene expression

how can gene expression be measured?

probes

what is a molecular probe?

a molecule complementary to a target sequence

• for DNA → labelled ssDNA binds to denatured DNA sample

how are DNA probes labeled?

polynucleotide kinase phosphorylates the $\lambda$ phosphate of ATP to the free hydroxyl at the 5’ end of the single strand

how can PCR be used to make labelled DNA probes?

• PCR run with labelled dNTPs

• PCR product denatured

describe the process of blotting

• cut DNA/RNA with restriction enzymes

• run on agarose gel

• denatured by an alkaline solution

• transferred to a membrane

• hybridize the membrane with probes and wash to remove other bands

what is different about RNA blotting?

• ssRNA forms a lot of secondary structures

• must be denatured before loading onto gel

what is the difference between southern blots, northern blots, and western blots?

• southern → DNA

• northern → RNA

• western → protein

what can a southern blot tell about a DNA sample?

• whether a gene is present in a sample or not → DNA fragment containing the gene of interest will take on the probe

• gene mutations which alter restriction sites will result in variations of fragment length

what can a northern blot tell you about an RNA sample?

• whether a gene is being expressed by a certain cell

• intensity of the band → how much the gene is being expressed

• band position → different sizes of RNA fragments indicate alternative splicing/mRNA modifications

what is qPCR used for?

used to measure DNA quantity

what is the process of qPCR?

• PCR with the incorporation of a fluorescent dye

• measure fluorescence in realtime

• samples with more starting DNA will show a certain level of fluorescence before one with less

how are cDNA libraries made from mRNA?

• mRNA poly-A tail primed with a poly -T oligonucleotide

• RT uses the poly-T primer to make a single stranded cDNA

• RNaseH removes RNA

• polydG adapter added to 3’ end

• polydC primer initiates replication of second strand

what is the process of microarray analysis of cDNA libraries?

• extract mRNA from two samples → RT makes cDNA with different fluorescent labels

• hybridized to a microarray containing many probes

• color and intensity at each probe will tell how much the gene is being expressed in each sample

describe the process of RNA-seq

• RNA → cDNA via RT

• sequencing of cDNA fragments

• tells you how many sequences correspond to

what are the differences between northern blots, RT-qPCR, microarray analyses, and RNA-seq?

• northern → detect size and abundance of a known mRNA transcript in a sample

• RT-qPCR → compare level of a gene’s expression in multiple samples

• microarray → measure the abundance of many genes in a single mRNA sample

• RNA-seq → good for revealing transcripts that don’t have probes

where does transcription start? what sequences surround it?

transcription initiation site (+1)

• coding sequence downstream

• promoter sequence upstream is where RNA polymerase binds

what are the stages of transcription?

• initiation → RNA pol binds to the promoter

• elongation → base pairing

• termination → RNA pol detaches from template

does RNA polymerase always start where it should? how is this solved?

no

• $\sigma$ factors allow it to bind specifically to promoters

• DNA binding proteins bound to the promoter enhance or impede

how many proteins are coded for in prokaryotic vs eukaryotic genes?

• prokaryotes = polycistronic → multiple proteins

• eukaryotes = monocustronic → one protein

how sensitive is each RNA polymerase to $\alpha$ -amanitin?

• RNA pol I → insensitive

• RNA pol III → very sensitive

• RNA pol II → moderate sensitivity

what types of RNA are transcribed by each RNA polymerase?

• RNA pol I → rRNA

• RNA pol II → mRNA, snRNA, siRNA, miRNA

• RNA pol III → tRNA and other rRNAs

what feature distinguished eukaryotic RNA polymerase from prokaryotic?

eukaryotic RNA polymerase has a clamp domain that allows it to stay bound to DNA while transcribing long genes → possessive

what feature distinguished RNA polymerase II from the other two?

the presence of the unstructured carboxy-terminal domain (CTD)