Restriction Enzymes

Radioactive phosphate

How will recognize the probe in hybridization? a) radioactive phosphate b) radioactive hydrogen

X-gal

If you use a substrate similar to lactose, called ___, then the bacteria will turn blue as they digest it. a) ligase b) X-gal c) LacZ

Endonucleases

Restriction ____ are special enzymes that bind to specific short sequences of DNA and make a cut. a) nucleus c) DNA c) endonucleases

Will not

Screening Process: First you have to find the clones that have taken up the vector. If the bacteria did not take up the vector, it _____ be resistant to ampicillin, and they ____ grow. a) will b) will not

inserted

Screening Process: Second, you have to find the clones that did take up a vector and has human DNA ____ into the multiple cloning site. a) deleted b) inserted

DNA ligase

Sticky ends make possible recombination of fragments generated by cut restriction enzymes. Fragments with the same sticky ends could pair up and be resealed by _____. These DNA fragments can be from different species. a) DNA ligase b) RNA polymerase c) palindromes

True

True or False: Cloning a Gene - 1. Extract and purify DNA (from human and bacteria) 2. Digest human DNA with restriction enzymes 3. Cut plasmid (bacterial DNA) with the same restriction enzyme 4. Insert DNA into plasmid (The enzyme ligase glues DNA together) 5. Make bacteria take up DNA (remember 3 ways) 6. Grow bacteria overnight to make millions of copies 7. Spread bacteria on a culture dish to separate individual clones to work with your gene.

LacZ

___ is one of the genes that code for a protein that breaks down lactose. a) ligase b) X-gal c) LacZ

DNA library

____ is a collection of DNA fragments representing all of the DNA from an organism. There will be thousands of human DNA fragments in the bacterial plasmids of a _____ to sort through. a) DNA dictionary b) DNA library

Hybridization

____ is a common method that uses a probe consisting of a complementary nucleic acid sequence to that of the gene of interest. 1. a) Hybridization b) Cloning

Bacteria

______ can use restriction enzymes to fight viral infections, and they can also select for gene sequences in strands that they take in through transformation. Protect own DNa from restriction enzymes with methyl groups (methylase enzymes block recognition sites). a) Eukaryotes b) prokayoyes c) bacteria

Plasmids

A type of vector: Bacteria are easy to grow and will easily take up _____ by a process called transformation. Many ____ have been engineered to contain an origin of replication, a selectable marker, which is usually an antibiotic resistance gene. Many restriction enzyme cut sites that are usually placed into a multiple cloning site (MCS) that are sometimes found within another selectable marker, such as the lacZ gene. a) plasmid b) bacteriophages c) Cosmids d) YACs (Yeast artificial chromosomes)

Bacteriophages

A type of vector: ____ are viruses that infect bacteria. All of the lambda phage can be used to carry gene to a bacterium. The genes involved in the lysogenic phase can be removed and replaced with foreign DNA without affecting the ability of the phage to infect bacteria and replicate. The phage containing recombinant DNA can then infect bacterial cells, process called transduction. a) plasmid b) bacteriophages c) Cosmids d) YACs (Yeast artificial chromosomes)

Cosmids

A type of vector: ____ laboratory constructed vectors that contain the cos site from the lambda phage, which is required for packaging chromosomes into virus particles. They contain plasmid features such as antibiotic resistance and an origin of replication. ____ are used to carry much larger sequences of DNA than plasmids. a) plasmid b) bacteriophages c) Cosmids d) YACs (Yeast artificial chromosomes)

YACs

A type of vector: _____ are beneficial because these vectors can be grown in yeast cells, which are eukaryotic. Since prokaryotes lack the ability to process RNA, a eukaryotic system is very beneficial. ____ have an origin of replication, and a centromere so that DNA can be distributed during mitosis, restriction enzyme cut sites, and yeast-specific selectable markers. Yeast cells are easily grown and have been used to express many mammalian genes. a) plasmid b) bacteriophages c) Cosmids d) YACs (Yeast artificial chromosomes)

Vectors

All ____ must have certain characteristics in order for them to be useful to genetic scientists: 1. They must be able to replicate within the host cell 2. They must contain restriction enzyme cut sites 3. They should carry a selectable marker to allow for identification of the host cells that contain the recombinant DNA 4. The vector should be easy to recover from the host cells that contain it. a) vectors b) sticky ends c) palindromes

Insert

The MCS (Multiple Cloning Sites) - Know that there is a "start" site for transcription. Know that there are multiple restriction enzymes sites where you can ____ your gene a) delete b) insert

White

The colonies that are ____ will have recombinant DNA. a) white b) blue

Sticky ends

The cut made by restriction enzymes is also unusual. The cut is not necessarily made in the center of the sequence but to one side. This creates a break with short single strands of DNA dangling from each end. These overhangs are called ___. The same ___ are always produced from cuts with the same restriction enzymes. a) dangles b) sticky ends c) hangovers

Palindromes

These sequences are symmetrical in that the DNA double helix has identical sequence running in opposite directions called ____ a) palindromes b) helicases

True

These short sequences are usually 4-6 nucleotides long

True

True or False: Hybridization 1. Grow previously screened clones on agar plates - with no ampicillin to prevent the growth of bacteria with no plasmid - bacteria must be spread on plate sparsely so that each colony will be identical (clones) 2. Press a special filter paper onto the colonies to create a replica of some of the cells. 3. Treating the filter paper with a solution to denature the DNA into single strands 4. Wash the filter in a solution of radioactively-labeled probe (the probe only hybridizes with DNA from colonies that contain the gene of interest because we made a probe that would only bind to the insulin gene (probes are usually about 20 nucleotides long) 5. The filter with hybridized DNA is overlaid on photographic film. (Any radioactivity will be revealed as a black spot on the film) 6. The position of the black spot can be compared to the original master plate of colonies (the colonies containing the genes are then identified) 7. Then you can choose only bacterial cells from that colony and grow them in large quantities - then isolate insulin protein made by the bacteria.

True

True or False: If your gene inserted into the MCS, the LacZ gene will be disrupted and the bacteria will not turn blue, they will be white.

True

True or false: Cloning genes: There are three ingredients necessary for genetic engineering. 1. a source of DNA that contains the gene you want to transfer - think of the human insulin example. 2. One or more restriction enzymes to cut the DNA 3. A vehicle (called a vector) to carry the source of DNA into the host cell

Binary fission

Why use bacteria? They are easy to grow. They replicate your gene when they replicate their own DNA before they divide by _______. They reproduce themselves by the millions overnight - therefore you can make many copies of your gene in 1 day. a) replication b) binary fission