6: DNA Technology

what DNA technology…

• amplifies specific DNA sequences from a minute amount of starting material

• requires prior knowledge of the target sequence

• reveals whether the sequence is present or not

polymerase chain reaction (PCR)

what diagnostic method works by…

• separating dsDNA using heat

• binding DNA primers to a target sequence during annealing

• synthesizing new DNA with DNA polymerase

polymerase chain reaction (PCR)

which DNA technology would you use when…

• the DNA target sequence is known

• rapid results are needed

• diagnosis depends on detecting a specific pathogen, mutation, or allele

polymerase chain reaction (PCR)

which DNA technology…

• quantifies the amount of a DNA target by using fluorescent dyes or probes during amplification

• used in clinical labs to monitor viral load and treatment responses

quantitative polymerase chain reaction (qPCR)

which DNA technology works by…

• converting RNA to cDNA using a reverse transcription step

• uses quantitative PCR to amplify and measure the amount of the original RNA

reverse transcription quantitative polymerase chain reaction (RT-qPCR)

which diagnostic method is considered the gold standard for RNA viruses like SARS-CoV-2 and HIV and is used when quantification of DNA or RNA is clinically meaningful?

reverse transcription quantitative polymerase chain reaction (RT-qPCR)

which DNA technology works by…

• fixing patient cells on a slide

• denaturing the DNA

• adding fluorescently labeled probes that bind to complementary sequences

• uses a fluroesence microscope to view signal patterns that indicate normal or abnormal chromosome structure

fluorescence in situ hybridization (FISH)

which DNA technology would you use when a specific chromosomal abnormality — like a translocation, gene amplification, or deletion — is suspected?

fluorescence in situ hybridization (FISH)

which DNA technology is used to detect copy number variations?

Array-CGH (DNA Microarray)

which diagnostic technique works by…

• extracting and labeling patient and reference DNA with different fluorophores

• mixing and hybridizing them to a microarray slide

• using a computer to measure fluorescence to detect duplications or deletions

Array-CGH (DNA Microarray)

which DNA technology would you use when…

• large deletions or duplications are suspected

• higher resolution that karyotyping is needed

Array-CGH (DNA Microarray)

which DNA technology is used…

• to determine the exact nucleotide sequence of a small, known DNA region using the “chain termination” technique

• often used to confirm a variant identified by other screening methods

sanger sequencing

which sequencing method involves…

• amplifying a DNA region via PCR

• adding fluorescently labeled dideoxynucleotides (ddNTPs) that terminate synthesis

• separating the resulting fragments by capillary electrophoresis to read the sequence

sanger sequencing

which DNA technology would you use when you need to confirm a known mutation in a specific gene or exon, such as the ΔF508 mutation in cystic fibrosis?

sanger sequencing

which DNA sequence allows millions of DNA fragments to be sequenced in parallel to provide single-nucleotide resolution across large genomic regions?

next generation sequencing (NGS)

which sequencing technique works by…

• fragmenting DNA

• ligating it to adaptor sequences

• amplifying clusters of identical fragments

• adding fluorescently labeled dNTPs one base at a time

• aligning the resulting reads to a reference genome to identify mutations

next generation sequencing (NGS)

which DNA technology would you use when…

• the causative mutation is unknown

• many genes or the whole genome needs to be searched to find SNPs, indels, CNVs, or gene fusions

next generation sequencing (NGS)

what key principle states that single-stranded DNA or RNA will bind to a complementary sequence under the right conditions?

DNA and RNA hybridization

what is the difference between PCR and qPCR

PCR = size-specific and qualitative

qPCR = not size-specific and quantitative