Methods_01

Oligo A, because of the higher GC content

Yes, 38% G + 38% C + 12% A + 12% T

DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue.

Alkaline lysis

- Grow bacteria overnight

- Collect cells by centrifugation

- Resuspend the cells in a buffer (contains glucose to maintain osmolarity, EDTA to chelate and de-stabilize membrane proteins, and Tris to maintain pH)

- Lyse the cells with SDS (a detergent that makes holes in the membranes) plus NaOH (loosens cell walls, denatures the proteins, releases and denatures DNA)

- Neutralize the solution with potassium acetate that converts SDS to insoluble KDS: at neutral pH plasmid DNA re-anneals, whereas sheared chromosomal DNA remains single stranded and insoluble at high salt

- Centrifuge the sample to get rid of cellular debris, denatured proteins, KDS, and ssDNA chromosomal DNA

- The supernatant that contains plasmid DNA can then be either passed through a silica membrane (that will bind plasmid DNA under high salt conditions) or precipitated with alcohol and centrifuged to collect the DNA on the bottom of the tube

- Plasmid DNA (on a membrane or in a pellet) is washed with 70% ethanol

- Pure DNA (eluted from the membrane under low salt conditions or resuspended from a pellet) can then be used for downstream applications (PCR, restriction digest, etc.)

The multiple cloning site in a plasmid contains recognition sequences for different restriction enzymes. Enables a few different ways to clone into the vector

Restriction enzymes bind to dsDNA and cut phosphodiester bonds in both strands of the DNA double helix

5' and 3' sticky ends, blunt ends

• DNA Ligase

Subcloning refers to cutting and ligating desired pieces of DNA together in a vector

E.coli cells can be made 'competent' to accept extraneous DNA

Chemically competent cells are prepared by re-suspending cells in a CaCl2 solution (which permeabilizes E. coli cell membranes) and subjecting the cells to a mild heat shock (~42^oC)


Electrocompetent cells are prepared by washing the cells with water to remove salts and subjecting the cells to a short electric pulse

Convey it some evolutionary advantage. Plasmid might have Ampicillin resistance to convey to the bacteria, when it is grown on a me

An AT-rich region of DNA where replication starts

Determines plasmid copy number by controlling how often the plasmid replicates

Plasmid replication may rely on the host replication machinery or be controlled by plasmid-encoded replication factors (Rep proteins) and RNA

Accordingly, plasmid replication can be dependent or independent of chromosomal DNA replication and host cell cycle

Presence of a reporter gene facilitates selection of recombinant plasmids (as opposed to the re-ligated parentalplasmids)

The LacZ gene encodes an enzyme (beta-galactosidase) that converts a colorless substrate X-gal into a blue product

If a piece of foreign DNA is inserted into LacZ, the LacZ gene is disrupted, and no enzyme is made, so the colonies harboring the recombinant plasmid are white


This allows to distinguish recombinant (white) clones from empty (blue) clones

• Maxam-Gilbert chemical modification and cleavage
• Sanger dideoxy chain-termination

negatively charged DNA flows to the positive pole smaller DNA fragments travel faster allowing the different sized fragments to be separated

1. Perform a restriction digest on the DNA from the white colony and run the products on a gel (restriction digest + gel electrophoresis)

2. Run a PCR using gene-specific primers

3. Sequence the insert and its junction with the vector

directional PCR- would only amplify gene if the primers chosen work only when the insert is in the corect location

Sanger sequencing can give you the code to see if its correct orientation

Restriction mapping can give you size information to let you know if insert is present/in correct position

EtBr (dye), GelRed, SYBR, GelGreen

a dye commonly used to visualize DNA on a gel.

EtBr is an intercalating agent that inserts itself between the stacked base pairs of DNA

EtBr-bound DNA glows under UV light, making it possible to visualize DNA fragments on a gel

(Dr. Stepanova's previous generation used to put it in their mouth for halloween)

Lanes 2 and 5

Sequence the insert and its junction with the vector

In a freshly cut vector, assume all 5' phosphates are present.

De-phosphorylate the vector with alkaline phosphatase.

T4 DNA ligase plus the insert adds the insert to the vector

alkaline phosphatase

Ligation of the blunt ends may be less efficient

Two blunt ends may indicate compatibility, but there is no directional cloning

Vector self-ligation is high

Use 2 different restriction enzymes to make different sticky ends around your insert genes to make a directional insert.

Set up a PCR reaction using a forward vector primer plus a reverse insert-specific primer (or a forward insert-specific primer plus a reverse vector primer)

Primers will only amplify if the insert is present and is in the right orientation

Polymerase chain reaction

PCR is used to selectively amplify multiple copies of the DNA fragment of interest

Choose an enzyme or pair of enzymes that cuts in the vector and asymmetrically within the insert

- DNA template  

- DNA polymerase  

- DNA primers* (F+R)  

- dNTPs  

- Buffer with Mg++

no in vivo cloning/transformation/colony picking necessary

array based sequencing gives a higher degree of parallelism than capillary-based sequencing

Genomic DNA is fragmented, then cloned to a plasmid vector and used to transform E. coli. For each sequencing reaction, a single bacterial colony is picked and plasmid DNA isolated. Each cycle sequencing reaction takes place within a microliter-scale volume, generating a ladder of ddNTP-terminated, dye-labeled products, which are subjected to high-resolution electrophoretic separation within one of 96 or 384 capillaries in one run of a sequencing instrument. As fluorescently labeled fragments of discrete sizes pass a detector, the four-channel emission spectrum is used to generate a sequencing trace.

• Common adaptors are ligated to fragmented genomic DNA, which is then subjected to one of several protocols that results in an array of millions of spatially immobilized PCR colonies or 'polonies’.
• Each polony consists of many copies of a single shotgun library fragment. As all polonies are tethered to a planar array, a single microliter-scale reagent volume (e.g., for primer hybridization and then for enzymatic extension reactions) can be applied to manipulate all array features in parallel.
• Similarly, imaging-based detection of fluorescent labels incorporated with each extension can be used to acquire sequencing data on all features in parallel. Successive iterations of enzymatic interrogation and imaging are used to build up a contiguous sequencing read for each array feature.

• 1. Library preparation
• 2. Clonal amplification
• 3. Cyclic array sequencing

Pooled amplicons are clonally amplified in droplet emulsions. Isolated DNA-carrying beads are loaded into individual wells on a PicoTiter™ plate and surrounded by enzyme beads. Nucleotides are flowed one at a time over the plate and template-dependent incorporation releases pyrophosphate, which is converted to light through an enzymatic process. The light signals, which are proportional to the number of incorporated nucleotides in a given flow, are represented in flowgrams that are analyzed and a nucleotide sequence is determined for each read with the GS Amplicon Variant Analyzer software.

Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily automated.

Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and gel-electrophoresis.

DNA polymerase adds nucleotides and releases PPi

Sulfurylase regenerates ATP (out of APS and PPi)

Luciferase hydrolyzes ATP in the presence of its substrate luciferin, producing light

Apyrase hydrolyzes/inactivates extra nucleotides (not incorporated in the DNA)

The first few steps, including emulsion PCR on the beads, are the same as in 454, but 3’ ends of the PCR fragments are modified to enable their covalent attachment to a glass slide

Beads are deposited onto glass surface and the sequence of the DNA fragments they carry is determined using a set of 16 colored “interrogation probes”

Each probe is an octamer, made of (3′-to-5′ direction!) 2 probe-specific bases and 6 degenerate bases (nnnzzz) with one of 4 fluorescent labels linked to the 5′ end

The 2 probe-specific bases in the 3’ represent one of 16 possible 2-base combinations

• When a nucleotide + DNApolymerase adds the nucleotide to the DNA backbone, pyrophosphate and H+ is released. In 454 Pyrosequencing can detect the release of pyrophosphate and through enzymatic activity, light is released to be used as an indicator to which nucleotide was added.

4 of 16 probes are added at a time