DNA Sequencing (9/6-9/11)

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Sanger DNA Sequencing

Determination of the sequence of nucleotides in a particular piece of DNA

Sanger Sequencing Output (Hint: 2)

700-800 nt of a single molecule

Highly accurate (>99%)

Sequencing by Synthesis

Determine the sequence by synthesizing a strand of DNA

Template

The piece of DNA to be sequenced

Primer

A short (18-22 nucleotides) single stranded piece of DNA that is complimentary to one strand of the template

Oligonucleotide

Another name for a primer

DNA Polymerase (Hint: 2)

Catalyzes the addition of dNTPs to the 3’-OH of the growing daughter cell

Synthesizes DNA in the 5’ → 3’ direction only

Di-deoxy nucleotides (ddNTPs) (Hint: 2)

Block DNA synthesis; Lack an -OH group at the 3’ position

DNA polymerase cannot add a nucleotide to a ddNTP

Chain Termination (Hint: 2)

Another name for Sanger Sequencing

A technique for DNA sequencing based upon the selective incorporation of chain-terminating dideoxy nucleotides (ddNTPs) by DNA polymerase

Read

The sequence of a single molecule

Shotgun Sequencing

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

Genomic Library (Hint: 3)

Genome is sheared and cloned randomly into plasmids which are transformed into E. coli

Individual colonies are selected the plasmid purified, and the genomic fragment sequenced

Sequence overlaps are assembled into the genome by computer algorithms

Illumina Sequencing (Hint: 5)

Generated by ligating sequence specific adaptors

Individual molecules made into clusters by bridge amplification

400 million 300 bp reads (short)

Total Output: 120 Gb

Highly accurate (>99.9%)

PacBio Sequencing (Hint: 10)

Real-time sequencing by synthesis produces long reads

Fluorophore clipped off by polymerase

DNA synthesized is natural

No steric hindrance or accumulation of background signal

All four nucleotides added at once

Up to 8 million reads

10-15 kb average read length (long)

Total output: Up to 200 GB

Low single read accuracy (86%)

Consensus read accuracy >99%

Oxford Nanopore Sequencing (Hint: 5)

Detects electrochemical differences between the base pairs passing through a small protein pore in a membrane

10-15 kb average reads with some ultra long reads (>100 kb)

Total output is unlimited

Low single read accuracy (85%)

High consensus accuracy (>99%)

RNA-seq

Sequence all of the RNAs expressed in a cell

Transcriptome

The collection of RNA transcripts in a cell, tissue, or organism

ChiP-seq (Hint: 2)

Chromatin Immunoprecipitation

Identify protein binding sites in entire genome

DNA Sequencing

A laboratory technique that determines the order of the four bases that make up DNA

How is the primer labeled?

Labeled with ³²P at the 5’ end

How many reactions are used to do chain termination? What is different in each reaction?

4 sequencing reactions

What ddNTP is added

How are the fragments produced in Sanger sequencing separated and visualized? (Hint: 3)

Polyacrylamide gel electrophoresis is used to visualize the results of the sequencing reaction

Denature the DNA

Run each reaction in a separate lane

How do you read a sequencing gel?

From the bottom

How is automated Sanger sequencing different from classic Sanger sequencing? (Hint: 4)

All 4 reactions carried our in one tube

Use ddNTPs fluorescently labeled with 4 different colors

Sequence of colored bands is detected automatically with a laser

Produces a sequence trace file from which you can call bases and build the sequence

Where is sequencing data stored in the United states?

GenBank

Housed at the National Center for Biotechnology Information (NCBI)

What is the Illumina sequencing cycle? (Hint: 5)

Add 4 labeled nucleotides and DNA polymerase

3’ Reversible terminator ensures that only 1 nucleotide is added per cycle

Take thousands of pictures across the flow cell

Call the nucleotide incorporated at each spot

Remove terminators and dyes