DNA Sequencing

What is DNA sequencing?

the lab process of determining the exact order of the four nucleotide bases in DNA

What does sequencing-by-synthesis mean?

a method that reads the genetic code by tracking the addition of nucleotides as a new DNA strand is copied

What is the template

the piece of DNA to be sequenced

What is the primer?

a short, single-stranded piece of DNA that is complementary to the one strand of the template

What is an oglionucleotide?

a short, single-stranded or double-stranded chain of DNA or RNA molecules

How is a primer labeled?

the primer is labeled 32P at the 5’ end

How does DNA polymerase synthesize DNA?

is synthesized DNA in the 5’-3’ direction only

What strand of DNA is sequenced in Sanger sequencing?

semi-conservative DNA (single-stranded)

What is a ddNTP?

dideoxyribonucleic acid

How are ddNTP’s used to do chain determination?

they lack the 3’ hydroxyl which make it impossible for the DNA polymerase to attach to

How many reactions are used to sequence?

four separate reactions

What is different in each type of reaction?

the type of ddNTP added to each tube

How are fragments produced in Sanger sequencing separated and visualized?

DNA is denatured and then run on polyacrylamide gels and is exposed using X-ray film

How do you read a sequencing gel?

From the bottom up, 5’-3’

How is automated Sanger sequencing different from classic Sanger sequencing?

automated uses single-tube fluorescent labeling and laser detection

Where is sequencing data stored in the United States?

in public federal data bases funded by tax dollars

What is shotgun sequencing?

random sequencing of small portions of a genome and reassembly by overlapping

How are genomic sequences produced by shotgun sequencing?

larger strands of DNA are broken into smaller strands, and then computers assemble the genome

What is a genomic library?

genome is shared and cloned randomly into plasmids, which are transformed into E. Coli

What is next-generation or high-throughput sequencing?

technology that determines the order of nucleotides in DNA or RNA very quickly

how does Illumina sequencing work?

reads millions of DNA pieces at the same time by copying them one step, or one base, at a time

What is the Illumina sequencing cycle?

one single nucleotide is read at a time on a growing strand

How much data can be produced?

400 million 300 bp

What is PacBio sequencing?

real-time sequencing that produces long reads

How is PacBio sequencing similar to and different, from other sequencing techniques?

• It is sequence-by-synthesis like Illumina

• It adds all four nucleotides at once, which is different

In principle, how does PacBio sequencing work?

the fluorophore is clipped off by the polymerase

What is Oxford nanopore sequencing?

a method that directly reads single molecules of DNA or RNA by tracking changes in electrical currents

How is Oxford nano sequencing similar to and different from other sequencing techniques?

• it is NOT sequence-by-synthesis

• it detects electrochemical differences between base pairs

In principle how does Oxford nanopore sequencing work

by passing through a small pore in the membrane

What is RNA-seq?

sequences all of the RNAs express in a cell

What is the transcriptome?

complete collection of all RNA transcripts produced in a cell of a specific tissue type at a given time

What is ChIP-seq and what does it identify?

chromatin immunoprecipitation that identifies protein binding sites in an entire genome