Nucleic Acids and Recombinant DNA Technology

Flashcards covering the final exam format, basic cloning processes, restriction enzymes, plasmid vectors, cDNA/genomic libraries, and DNA analysis techniques like Southern blotting based on the Chapter 41 lecture notes.

What is the format of the final exam according to the lecture notes?

The final exam consists of 65 questions (out of 60) with the following content breakdown: Proteins (30%), Carbohydrates (10%), Lipids (10%), and Nucleic Acids (50%).

According to the transcript, what are seven applications of DNA technology?

1. Production of large quantities of recombinant human proteins (e.g., insulin, human growth factor). 2. Genetic modifications to agricultural products. 3. Forensic medicine (identification of criminals, relatives, or victims). 4. Prenatal diagnosis (e.g., sickle cell anemia, cystic fibrosis). 5. Identification of genetic diseases (e.g., breast cancer). 6. Basic science studies (protein sequencing, gene mapping). 7. Potential applications in biological warfare.

How is reverse transcriptase used in the context of proinsulin mRNA?

Reverse transcriptase is used to transcribe isolated proinsulin mRNA into cDNA ($complementary DNA$).

What are the six basic steps in a recombinant DNA experiment?

1. Preparation of DNA (vector and target). 2. Cleavage of DNA at particular sequences using restriction enzymes. 3. Ligation of DNA fragments. 4. Introduction of recombinant DNA into compatible host cells (genetic transformation). 5. Replication and expression of recombinant DNA in host cells. 6. Identification of host cells containing the recombinant DNA of interest (screening and selecting).

What is a plasmid and what are its key roles in cloning?

A plasmid is an independently replicating DNA circle. In cloning, foreign DNA is inserted into it for replication. Plasmids typically carry drug resistance genes used for selection and can spread antibiotic resistance between bacterial species.

What are restriction enzymes and how do they function?

Restriction enzymes are bacterial endonucleases that cleave DNA at specific palindromic sequences (4 to 8 nucleotides long). They protect bacteria against foreign DNA, such as bacteriophages, while the bacteria's own DNA is protected by methylation.

What are the specific recognition sequences for the restriction enzymes EcoR1 and BamH1?

EcoR1 cuts at $GAATTC$ and BamH1 cuts at $GGATCC$.

What is the difference between 'selection' and 'screening' in cloning?

'Selection' determines if the plasmid was taken up by the cell (typically via antibiotic resistance), while 'screening' determines if the gene of interest was successfully cloned into the plasmid.

What are the essential components for a plasmid to replicate and be useful as a cloning vector?

It must be circular and contain a replicon or origin of replication ($ori$) for DNA polymerase binding. It also requires a selectable marker (drug resistance) and a multiple cloning site ($MCS$) or polylinker.

What is a 'palindrome' in the context of restriction sites?

A palindrome is an inverted repeat where the sequence reads the same in the $5'$ to $3'$ direction on both strands.

What are the advantages and disadvantages of using Genomic DNA for cloning?

The advantage is that it covers the whole genome (you get everything). The disadvantage is that it contains introns which cannot be recognized by hosts such as $E. coli$.

What is a cDNA library and how is it purified?

A cDNA library represents all mRNAs made in a specific cell or tissue, created as double-stranded DNA via reverse transcriptase. Purification of mRNA for the library relies on the polyA tails of mature eukaryotic mRNA.

Why must mammalian genes be converted to cDNA for expression in bacteria?

Mammalian genes contain introns and exons; because bacteria cannot process introns, researchers must prepare cDNA which consists only of the expressed, mature DNA sequence.

In the blue/white screening system, what does the color of the colony indicate?

Blue colonies indicate cells transformed with cloning vectors that do not have the insert (eta ext{-galactosidase} is active). White colonies indicate the insert is present, as it disrupts the eta ext{-galactosidase} gene.

How does X-gal produce a blue pigment?

X-gal is an analog of lactose hydrolyzed by eta ext{-galactosidase} to form $5 ext{-bromo-4-chloro-indoxyl}$, which then dimerizes to produce the insoluble blue pigment $5,5 ext{’-dibromo-4,4’-dichloro-indigo}$.

What is the Southern blot and what are its steps?

Developed by Edwin Southern, it identifies DNA with specific base sequences. Steps: 1. Digest DNA with restriction endonuclease. 2. Electrophoresis to separate fragments. 3. Blot fragments onto a nitrocellulose membrane. 4. Hybridize with a $^{32}P$ probe. 5. Perform autoradiography.

What are the characteristics of the plasmid vector pBR322?

It has $4361$ base pairs, an origin of replication ($ori$), and carries antibiotic resistance genes for ampicillin ($amp$) and tetracycline ($tet$).

What are the two levels at which a clone of interest can be examined after transformation?

It can be examined at the transcriptional level (probing for cDNA) or the translational level (probing for the protein of interest).