Digital Analysis of Genomes

The cell nucleus is too much material and information to study as a whole, what are two strategies researchers use to compartmentalize it?

What kind of enzymes are used to cut the genome at specific locations? How do they work?

Restriction enzymes originate in and can be purified from _______ cells. What is their function there?

How does the bacteria protect its genome from digestion by the restriction enzymes?

In the test tube, restriction enzymes from bacteria recognize target sequences of ______ base pairs in DNA isolated from any other organism and cut the DNA at or _____ these sites. How long are most recognition sites?

Explain the palindromic nature exhibited between restriction enzymes and restriction sites

How do the enzymes make their cuts?

Explain plating efficiency
What happens with the progeny

When a petri dish is coated with a continuous lawn of bacterial cells, an active viral infection can be observed as a visibly cleared spot, or ______, where bacteria have been _________. What does a plating efficiency of nearly 1.0 mean?

Restriction is rarely _______. Although lambda virus grown on E. coli K12 produces almost no _______ (the viruses infect cells but can't ______ inside them), a few viral particles inside a few cells do manage to _______. If their progeny are then tested on E. coli K12, the ______ ______ is nearly 1.0

The phenomenon in which growth on a restricting host modifies a virus so that succeeding generations grow more efficiently on the same host is known as ________

Studies following viral DNA after bacterial infection found that during restriction, the viral DNA is broken into pieces and _________. When the enzyme responsible for the initial breakage was isolated, it was found to be an ________, an enzyme that breaks _______ bonds in the viral DNA molecule, usually making ______-strand  cuts at a specific sequence in the viral chromosome

Why did researchers call them restriction enzymes?

A small percentage of viral DNA escapes digestion and goes on to generate new viral particles was modified by the addition of _______ groups during their _______ in the host cell. What are the enzymes that add those groups called?

The average distance between recognition sites of any length is dictated by which general formula?

What is the probability that a four-base recoginiton site will be found at a given position in a genome?
What if it was a six-base recognition site?
What if it was an eight-base recognition site?

How many DNA fragments are produced by a 4-base recognition in a human genome of 3 billion base pairs?

Summarize the info in the figure:

Why can't we use restriction enzymes for experiments that require the random cutting of DNA?

Random cutting of DNA can be achieved by subjecting the molecules to ______ _____, such as passing the sample through very _____ needles at _____ pressure or by _______ (define). These cause different parts of the DNA to be pulled in different directions leading to breakage of _______ bonds at ______ positions, leaving the DNA _________

What if researchers want to adjust the number of fragments of DNA?

The ends of the DNA fragments produced by mechanical shearing are sometimes _____ or they may have protuding SSDNA ends. Why aren't they considered sticky?

To analyze the DNA in a sample, the technique _______ (define) is employed.

Biologists use electrophoresis to ______ many different types of molecules, for example DNA of one _____ from DNA of other _____, DNA for _____ or one kind of _____ from another

How do we carry out such separations?

How are DNA molecules pulled through the gel toward the wire with a positive charge?

4 variables that determine movement of DNA toward the positive wire:
Which are constant and which aren't as DNA fragments migrate in a particular gel

With linear DNA molecules, differences in ______ are proportional to differences in _____: The longer the molecule the ______ the volume it occupies as a random coil. The _____ likely it is to make a ____ in the gel matrix big enough to squeeze through, and the more _____ it will bump into the matrix which will _____ its mobility

Long story short, ______ DNAs will travel greater distances from the wells than _____ DNAs

When electrophoresis is completed, the gel is incubated with a flourescent DNA-binding dye called _____ ______. After the unbound dye has been washed away, it is easy to visualize the DNA by placing the gel under an ______ ______ light, which causes the dye bound to the DNA fragments to glow in an _______ color. The sizes of the DNA molecules becomes apparent when you compare _______ ________ to those of known ______ fragments

Thoroughly study page 304

Molecular cloning

The two steps of molecular cloning
What does this have to do with libraries?

Small fragments of human genome ______ reproduce (replicate) on their own. So we must ______ each fragments into _______ which need which two specialized sequences?

The cutting and splicing together of vector and inserted fragment (DNA from two different origins) creates a _______ _____ _____

How do we make recombinant DNA molecules

The simplest vectors are minute circles of _____-stranded DNA known as ________ that can gain ______ to and ______ within many kinds of bacterial cells, _________ of the bacterial chromosomes

The most useful plasmids contain several ________ sites, one for each of several different _____ ______. This provides _____ in the choice of enzymes that can be used to _____ the DNA containing the ______ of interest.

Exposure to any of these restriction enzymes opens up the _______ at the corresponding _______ site, allowing the insertion of a _______ DNA fragment with the same enzyme, without at same time splitting the ______ into many pieces

Each plasmid vector carries an ______ ____ _______ and a gene for resistance to a specific _______. The origin of replication enables it to ________ ___________ inside a bacterium. The gene for antibiotic resistance confers on the host cell the ability to _____ in a medium containing a specific _______; the resistance gene thereby enables experimenters to select for propagation only those bacterial cells that contain a ______

Selectable markers

Plasmid vector restriction sites useful for cloning are ones that do not interrupt either the vector ______ _____ _______ or the ______ region of the ______ ______. How do plasmids increase the ease of purification?

Although each type of vector functions in a slightly different way, the general scheme of entering a host and taking advantage of the cellular environment to ______ itself is the same for all. How do scientists obtain E. coli cells that contain recombinant DNA molecules in which human DNA fragments were cloned into a plasmid vector? (2-story)

Transformation (define) of host cells

Under favorable conditions, what are the odds that one plasmid will enter the cells of E. coli vs two in the same cell?

To identify the the 0.1% of cells housing a plasmid, the bacteria-plasmid mixture is _______ onto a plate containing _____ nutrients, and _______. Only cells transformed by a plasmid providing resistance to _______ will be able to grow and multiply in the presence of the _______

Each viable ______-containing bacterial cell will multiply to produce a distinct spot on an _____ plate, consisting of a colony of tens of millions of genetically ______ cells. The colony as a whole is considered a ______ _____ while the millions of identical plasmid molecules conatined within a colony together make up a _____ ______

Moving step by step from the DNA of any organism to a single purified DNA fragment is a long and tedious process. Fortunately, scientists do not have to return to step 1 every time they need to purify a new genomic fragment from the same organism (explain how this is possible)

A perfect genomic library has one copy of every sequence in the entire genome. What is the formula for the # of clones in a perfect library?
Additionally, define a genomic equivalent

Explain why it is impossible to obtain a perfect library