Molecular Biotechnology Quiz

Recombinant DNA Technology

• Recombinant DNA technology uses two key tools: restriction enzymes and cloning vectors
• Restriction enzymes cut DNA at specific recognition sequences
• Cloning vectors are DNA molecules that accept DNA fragments and can replicate in host cells
• Cloning vectors must have several restriction enzyme sites to allow insertion of DNA fragments
• Cloning vectors also carry selectable markers to distinguish host cells that contain the vector with the cloned insert from those that do not.
• Cloning vectors often have a multiple cloning site (MCS) with many restriction enzyme sites
• Bacterial plasmids are commonly used as cloning vectors.
• DNA fragments can be linked using DNA ligase
• DNA ligase seals the phosphodiester backbone

Restriction Enzymes

• Restriction enzymes are produced by bacteria as a defense mechanism against bacteriophages
• Restriction enzymes cut DNA at specific recognition sequences.
• A palindrome is a DNA sequence symmetrically repeated on both strands
• Sticky ends (cohesive ends) have overhangs
• Blunt ends don't have overhangs

Palindromes

• Palindromes are nucleotide sequences that read the same on both strands
• Restriction enzymes recognize and cut DNA at specific palindromic sequences
• Sticky ends, generated by restriction enzymes, have complementary overhangs facilitating joining of DNA fragments via DNA ligase
• Blunt ends don't have overhangs and are easier to digest or introduce

Cloning Vectors

• Cloning vectors are DNA molecules that can accept DNA fragments
• They must be able to replicate independently of the host chromosome
• They have specific restriction enzyme sites to which DNA fragments can be added
• They also contain selectable marker genes to differentiate cells containing the recombined DNA molecules
• Plasmids are examples of commonly used cloning vectors.

Transformation

• Plasmids are introduced into bacteria via transformation
• Two main techniques are used:
• Using calcium ions and brief heat shock
• Electroporation: using electricity to move DNA into bacterial cells
• Cloning DNA involves cutting plasmid DNA and DNA to be cloned with the same restriction enzyme
• DNA restriction fragments are added to a linearized vector in the presence of DNA ligase
• Sticky ends anneal
• Recombinant DNA is produced and introduced into bacterial host cells by transformation

Blue-White Screening

• Not all plasmids incorporate DNA to be cloned; need to identify which plasmids have been recombined
• Selectable marker genes provide resistance to antibiotics (ampR) which help screen for useful recombinants from the transformation process
• Blue-white selection technique is used to identify cells with recombinants from nonrecombinants
• The plasmid contains the lacZ gene which encodes β-galactosidase
• Agar plates contain X-gal (analog of lactose), a substrate for β-galactosidase
• Cells with the functional lacZ gene and a nonrecombinant plasmid appear blue
• Cells with a recombinant plasmid and disrupted lacZ gene appear white

Other Types of Cloning Vectors

• Phage vectors are modified bacteriophage strains used for cloning
• Phage vectors can carry up to 45 kb of cloned DNA

Larger Cloning Vectors

• Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are used to clone large DNA fragments
• BACs are large plasmids with low copy number
• YACs have telomeres at both ends, an origin of replication (ori), and a centromere
• They can accommodate larger DNA inserts compared to plasmids

Expression Vectors

• Expression vectors are designed to ensure mRNA expression of a cloned gene
• Allows production of multiple copies of the encoded protein within the host cell.
• Plasmids, phage vectors, and YACs contain only DNA sequences and do not signal expression on their own
• Expression vectors can be used in both prokaryotic and eukaryotic host cells, such as in Ti plasmids and soil bacteria for use in plant tissue.

DNA Libraries

• Genomic libraries contain many overlapping fragments of the genome, each with at least one copy of every DNA sequence present in the organism's chromosomes.
• cDNA libraries represent only the genes expressed in a specific cell or tissue at a certain time, and therefore can be helpful in identifying genes involved in relevant processes.
• cDNA libraries are made by isolating mRNA from cells to synthesize complementary DNA (cDNA) using reverse transcriptase.

Reverse Transcriptase

• Reverse transcriptase utilizes mRNA as a template to synthesize cDNA
• This process results in an mRNA/cDNA duplex
• RNAse H partially digests the RNA component of the duplex
• This creates gaps in the RNA strand
• DNA polymerase I uses the 3' ends of the mRNA as primers to synthesize the second cDNA strand
• The result is a double-stranded cDNA molecule, which can be cloned or used for further analysis

Polymerase Chain Reaction (PCR)

• PCR is a rapid method of DNA cloning that extends the power of recombinant DNA technology.
• Eliminates the use of host cells. It copies a specific DNA sequence in vitro.
• Effective even when the target DNA sequence is present in very small amounts.
• PCR requires:
  • Double-stranded target DNA
  • DNA polymerase
  • Mg2+ (cofactor)
  • 4 deoxyribonucleoside triphosphates (dNTPs)
  • Primers (short, single-stranded sequences) PCR involves three steps:
  • Denaturation: dsDNA to ssDNA
  • Annealing: Primer annealing to ssDNA
  • Extension: DNA polymerase synthesizes new DNA strands using the primers and the separated strands as templates in successive cycles.

Molecular Techniques for Analyzing DNA and RNA

• Agarose gel electrophoresis separates DNA fragments based on size.
• Southern blotting is used to detect specific DNA sequences in a sample. It involves gel electrophoresis, transferring DNA to a membrane, and hybridizing the DNA with a labeled probe.
• Northern blotting analyzes gene expression by detecting RNA. It involves gel electrophoresis, transferring RNA to a membrane, and hybridizing the RNA with a labeled probe
• Western blotting is used for protein analysis. It involves separating proteins by gel electrophoresis, transferring them to a membrane, and detecting specific proteins using antibodies.
• Fluorescence in situ hybridization (FISH) method hybridizes DNA or RNA probes to chromosomes or tissue sections in situ

DNA Sequencing

• Sanger sequencing is the most common method for DNA sequencing. It utilizes dideoxynucleotides (ddNTPs) to terminate DNA synthesis at specific points during elongation.
• Computer-automated high-throughput sequencing significantly speeds up and increases the information obtained with Sanger. The process uses dideoxynucleotides that are labeled and thus have fluorescence to identify the next nucleotide that is incorporated.
• Next-generation sequencing (NGS) technologies allow for simultaneous reactions and massive amounts of DNA sequence data to be generated quickly with lower costs.

Gene Targeting and Knockout Animals

• Gene targeting involves introducing a targeted mutation into a specific gene in a genome
• Knockout mice are used to study the function of a specific gene by disrupting its function
• This is used to create embryonic stem (ES) cells that have the required mutation and homologous recombination.
• Results in nonfunctional proteins due to frameshift or other disruption to the gene.

Transgenic Animals

• Transgenic animals are created to express or overexpress a particular gene of interest.
• A vector with the transgene is introduced to ES cells. Recombination will put the transgene into the genome. -These are introduced to the developing embryo.
• Results in chimeras (part transgenic) or fully transgenic animals

Genome Editing with CRISPR-Cas

• CRISPR-Cas9 is a powerful genome editing tool discovered by scientists investigating how bacteria fight viral infections
• It enables the fast and efficient removal, insertion, and modification of DNA sequences.
• CRISPR-Cas relies on the nuclease Cas9
• Cas9 has double nuclease domains- it can double-strand break the DNA near the specific sequence, protospacer adjacent motif (PAM) 5' NGG 3'
• A guide RNA, sgRNA, directs Cas9 to target sequence, creating activity and specificity for the molecular process
• HDR can be tricked into using an artificial donor template for more precise genome editing using CRISPR-Cas.

CRISPR-Cas Applications

• CRISPR-Cas9 is used in various applications, such as creating tomatoes that ripen quickly, and combating livestock diseases.
• Gene therapy, a technique to treat genetic diseases, is another potential application of CRISPR-Cas9; clinical trials for cancer are also underway.