Recombinant DNA

Primer sequences are typically _____ nucleotides long

20-30

restriction enzymes cleave double-stranded DNA at the sites that show a particular type of symmetry; these sequences read the same on both star and and are called palindromes. Which of these sequences is not a palindrome? -5'-AGATCT-3' -5'-GGTACC-3' —5'CGGCCG-3' -5'CCTCAGG-3'

5'-CCTCAGG-3'

A southern blot is a technique that relies on hybridization of:

A nucleic acid probe to a complementary DNA

The transformation step in creating bacteria genetically engineered to produce human proteins involves:

Bacteria taking up the recombinant DNA in the form of vectors.

A graduate studen wants to create a recombinatn DNA molecule and introduce this molecule into bacteria. What is the proper order of steps that he should follow?

Choose plasmid/donor DNA -> cut with restriction enzymes -> join fragments via DNA ligase -> transform bacteria

One characteristic of restriction enzymes is that they cut:

Double-stranded DNA at specific sites.

True or false. If a restriction site of AATII is 5'-GACGTC-3' then 3'-GACGTC-5' is also an AATII resctrion site.

False. Bc sequence written from 3' to 5' should be complementary to the sequence written from 5' to 3', not identical to it. The correct sequence from 3' to 5' would be 3'-CTGCAG-5'.

How do the properties of DNA determine how it moves through a gel, is cut by restriction enzymes, and hybridizes to other DNA strands?

Fragments of DNA are negatively charged, so pieces will move through a porous gel when an electric current is passed through it. The distance that the DNA pieces move through the gel is based on their size, with larger fragments moving more slowly and smaller fragments moving through the gel quickly. to get these fragments of DNA, restriction enzymes, which each recognize a particular sequence of DNA (the restriction site), cleave the DNA at its specified site. In addition, DNA can hybridize to other DNA strands if their nucleotide sequences are complementary to each other.

How are DNA molecules sequenced?

In Sanger sequencing, the sequence of a template DNA strand is unknown. This DNA is used as the template for replication by DNA polymerase. A DNA primer, polymerase, normal nucleotides, and chain-terminating nucleotides are all added to the template and replication of the unknown strand begins. Elongation of this strand stops whenever a didexoynucleotide terminator ( a nucleotide in which the 3' hydroxyl group on the sugar ring is absent) is incorporated at the 3' end. The four altered nucleotides are chain terminators, each labeled with different fluorescent dye, so the result of Sanger sequencing is a tube filled with different-length fragments of the same DNA sequence. The mixture is then run on a gel and "read" by looking at the fluorescent pattern of the dideoxynucleotides incorporated into the sequence. For example, a sequence of 5'-ATGC-3' would be "read" as green-red-blue-purple (fo the fluorescent dyes).

A DNA molecule is cut with two different restriction enzymes known to cleave it only once each. After gel electrophoresis, three different DNA fragments are detected. This result means that the original DNA molecule was:

Linear. Bc digestion of a circular DNA molecule will produce only two DNA fragments.

What does PCR do? Name and explain its three steps, and escribe at least two uses for the PCR technique.

PCR is used to generate many copies of a piece of DNA. The three steps of PCR are 1. Denaturation of the double-stranded DNA into two individual strands, 2. Annealing of the two primers to their complementary sequence on the DNA template strands, and 3. Extension of the parental DNA strands through elongation (5' to 3') by DNA polymerase (by extending the primers). PCR can be used for a variety of purposes such as matching a person's DNA sequence to evidence found at a crime scene (paternity tests are performed in a similar way). OCR can also be used to indetify an organism based on a known conserved region of its DNA; it can alo be used to mass-produce certain sequences of DNA for DNA-based vaccines.

What is the name of the class of enzymes that recognizes and cuts a specific sequence of DNA?

Restriction enzymes

What does PCR require?

Template DNA, DNA polymerase, all four deoxynucleoside triphosphates, and two primers

In making recombinant DNA molecules that combine resctriction fragments from different oganisms, researchers usually prefer restriction enzymes such as BamHI or HindIII that generate fragments with "sticky ends" (ends with overhangs) rather than enzymes such as HpaI or SmaI that generate fragments with "blunt ends" (ends without overhangs). Can you think of a reason for this preference?

The advantage of sticky ends is that they give the researcher greater control over which restriction fragments can come together and be attached. Sticky ends can pair only with other sticky ends that have complementary 3' and 5' overhangs. Thus, restriction fragments produced by BamHI can combine only with other fragments produced by BamHI, and not with fragments produced by, for example, HindIII. However, any blunt end can be attached to any other blunt end; for example, a HpaI end can be attached to a SmaI end.

What features of DNA make it possible to make recombinant DNA in the lab?

The genetic code is the same for all organisms, Two double ehelices from different sources can be lighted together. Restriction enzymes cut DNA from all species.

How can recombinant DNA techniques be used to express a mammalian gene in bacteria?

The mammalian gene (donor DNA) can be expressed in a bacterium through its insertion in to a vector DNA that can be replicated in the bacterium. The same restriction enzymes are used to cut the donor DNA and the vector DNA so that they now have complementary ends. A ligation reaction is then performed to insert the donor DNA into the vector DNA. The vector DNA is then inserted into the bacterium and replicated through the normal method that also replicates the donor mammalian gene at the same time.

You have determined that the newly synthesized strand of DNA in your sequencing reaction has the sequence 5'-ACTACCGAGT-3'. What is the sequence of the template strand?

The sequence of the template strand is anti parallel to they synthesized strand and inferred from complimentary base pairing as 5'-ACTCGGTAGT-3'.

True or false. Restriction enzymes recognize certain DNA sequences and some of them will cut straight through, and others will leave an overhang at both ends of the cut.

True.

Which of the statements best describes the way you would engineer bacterial cells to produce a human protein?

Use a specific restriction enzyme to clave both the donor DNA and the vector DNA.

What is the polymerase chain reaction?

a technique for making copies of a piece of DNA, which allows a targeted region of a DNA molecule to be replicated or amplified into as mnay copies as diesired. It is both selective and higly sensitive.