nucleic acids: detection and quantification

qualitative analysis of gene products

• nature of molecule(s) in question

• size

• nucleotide composition

• conformation/configuration

• structure

quantitative analysis of gene products

determine levels of gene products

use of molecular probes

1. complex mixture of macromolecules binds to nitrocellulose or nylon membrane

2. probe specific for target binds

3. non-specific probe binding is removed

4. target is detected

polynucleotide kinase (PNK)

phosphorylates nucleotides by transferring the γ phosphate of ATP to the free hydroxyl at the 5’ end of the synthetic oligonucleotide with the reverse complementary sequence to known sequence containing gene of interest

α-phosphate

phosphate attached directly to nucleoside, incorporated into polymer

use of PCR to make labelled DNA probes

1. dNTPs with isotopic or covalently linked fluorescent label on α-phosphate are incorporated into PCR amplified DNA

2. unincorporated labelled dNTP substrates are removed

3. labelled PCR product must be rendered single-stranded before it can be used

analysis of nucleic acids by transferring to solid state support

• DNA is cut with a restriction enzyme then run through an agarose gel, producing a diagnostic signature

• mRNAs of different sizes will correspond to the various genes that encode them

• require that molecules be denatured

steps of DNA analysis by transferring to solid state support

1. DNA is cleaved with restriction enzyme

2. digested DNA fragments are filtered using gel electrophoresis

3. gel and nitrocellulose (solid state support) are sandwiched between filter papers and placed in alkaline solution

4. through capillary action, DNA is pulled out of the gel and deposited onto the nitrocellulose membrane

5. can be permanently bound by UV crosslinking

steps of RNA analysis by transferring to solid state support

1. RNA sample is denatured

2. denatured RNA is filtered using gel electrophoresis (maintaining denaturing conditions)

3. gel and nitrocellulose (solid state support) are sandwiched between filter papers and placed in denaturing buffer

4. capillary action transfers RNA from gel to nitrocellulose

5. can be permanently bound by UV crosslinking

UV crosslinked nitrocellulose (solid state support)

permanently records the levels (abundance) and position (size) of the molecules bound to the nitrocellulose membrane

hybridization of support (blot)

probes specific to target sequence binds, washes remove non-specific signal and only complementary sequences will be detectable on the blot following autoradiography

Southern analysis

nucleic acid probes with complementary sequence can bind nucleic acid targets, can be used to determine relatedness or for diagnostics

Northern analysis

nucleic acids of complementary sequences can base pair to form double stranded hybrids, enabling RNA detection, can be used to visualized tissue-specific or stage-specific (temporal) expression

quantitative PCR (qPCR) or RT-qPCR

• RT reaction converts all mRNA to cDNA

• PCR reaction includes an intercalating fluorescent dye that emits signal when incorporated into the growing DNA polymer

qPCR or RT-qPCR to determine mRNA levels

• plateau phase of PCR is reached must faster (in fewer cycles) in samples with greater amounts of starting material (cDNA)

• the amount of starting material can be calculated by monitoring the number of cycles it takes to reach the threshold cycle (C_t)

• more cDNA in original sample is directly proportional to the mRNA abundance in the original sample

making cDNA libraries

1. mRNA is converted to cDNA by priming poly A tail with single-stranded poly T oligonucleotide

2. RT uses this primer to initiate single-strand DNA synthesis that is fully complementary to the mRNA template

3. RNA is removed with alkali or RNaseH and poly dG adapter is annealed to the 3’ end

4. poly dC primer is used to initiate synthesis of the second DNA strand

5. E. coli DNA polymerase I progresses through any remaining hybrid regions and extends the second strand

RNA-seq

1. total RNA extracted from isolated cell or tissue population

2. specific RNA isolated by species (poly-A selection, size selection)

3. reverse transcriptase followed by RNase treatment and second strand synthesis converts RNA to cDNA

4. ligation of adaptors for NGS

5. PCR amplification and sequencing

6. genome alignment and quantification