Next Generation Sequencing and DNA Interactions Review

A comprehensive set of practice questions covering Next Generation Sequencing (NGS) methodologies, DNA-protein interaction mapping, DNA damage sequencing techniques, and CRISPR-Cas9 specificity analysis.

What is defined as 'read depth' in the context of sequencing?

Read depth refers to how many times a specific region of DNA was sequenced.

How does Taq polymerase facilitate cloning with small overhangs?

Taq polymerase can add an 'A' nucleotide without a template, making it easier to clone fragments with small overhangs.

What is the function of PNK (Polynucleotide Kinase) in DNA preparation for sequencing?

PNK adds a phosphate to the $5'$ end of the DNA in case it is missing.

Why is bridge amplification used during the sequencing process?

It is a PCR-like reaction that amplifies DNA at a specific location on the flow cell to provide a brighter fluorescent signal for reading.

How is the level of transcription determined using sequencing?

Different levels of RNA represent different levels of transcription; these are converted to cDNA and sequenced, where the number of reads correlates to the amount of RNA initially present.

In nuclease cleavage assays, what do regions with high levels of reads represent?

High levels of reads indicate where the protein was not bound (accessible DNA), whereas lower reads indicate where a protein was bound and blocked nuclease access.

What is the specific requirement for the Excision-seq technique to work effectively?

Cells must be highly irradiated with UV radiation to create enough DNA damage for the specific enzyme to target and fragment the DNA.

How does CPD-seq handle damaged DNA that cannot be ligated or sequenced?

The damage chemistry (such as CPD) must be reversed using recombinantly purified enzymes so that adapters can be added for sequencing.

What role does the 'seed region' play in CRISPR-Cas9 binding and cleavage?

The seed region is the PAM proximal region where complementarity is essential; mismatches here generally stop cleavage, while mismatches in the distal region are more tolerated.

What are the two possibilities for Cas off-target binding mentioned in the notes?

1. It could bind and cut the DNA. 2. It could bind and form a stable R-loop, blocking other cellular processes.

How are dsODNs (double-stranded oligodeoxynucleotides) used to find DNA breaks?

The cell is flooded with dsODN so that they insert themselves wherever a break occurs, allowing those specific locations to be amplified and sequenced.

What is the purpose of a Unique Molecular Identifier (UMI)?

UMIs are barcodes unique to every individual single DNA molecule, allowing researchers to track if a variant was present in the original molecule or was a mistake introduced during PCR.

What does the CHAMP (Chip-Hybridized Association Mapping Platform) method measure?

It uses an Illumina sequencing chip and TIRF microscopy to measure the DNA binding affinity ($K_d$) of a protein across thousands of different sequences simultaneously.

What are 'time barcodes' used for in pooled DNA library reactions?

Time barcodes are unique DNA sequences added at specific time points when a reaction is quenched to identify when a particular cleavage event occurred during sequencing.

How does decreasing positive charges on a guide RNA affect Cas9 specificity?

It reduces overall binding affinity, making off-target binding much worse while only slightly affecting on-target binding, thereby increasing the importance of guide RNA complementarity.