rDNA Techniques: Vectors and Enzymes Quiz

Purification of ____ Synthesis of first strand (cDNA)

Synthesis of second _____ Cloning Transcription

Exons Exons Exons Exons 1 2 3 4 _______

Introns Introns 1 Introns 2 1 2 1 3 4 3 2 ________

AAAA mRNA AAAA Intron splicing AAAA Processed ______

A cell expressing the required _____

DNA cloning involves combining nucleotide sequences from two different sources into the same DNA molecule in __________

Methods for making recombinant DNA are central to genetic __________

Sequencing of the human genome was largely completed by __________

A particular gene can be isolated and its function determined when a gene is inserted into a __________

Organisms can be 'engineered' for specific purposes like insulin production, insect resistance, etc., through the use of __________

Recombinant bacterium can be used to investigate protein/enzyme/RNA __________

Field inversion gel electrophoresis (FIGE) is a technique where the polarity of the electrodes is periodically ______ during electrophoresis.

Contour-clamped homogeneous electric field (CHEF) reorients the DNA at a smaller oblique ______, generally between 96 and 120 degrees, which makes it of use in DNA separation and analysis.

Excision of DNA fragments from a gel is a step in the process of ______.

Detection of DNA by Southern Blotting involves hybridization with a complementary radiolabeled ______.

The original method of blotting developed by Ed Southern in 1975 detects DNA fragments in an agarose gel that are complementary to a given nucleic acid ______.

The DNA on the membrane is fixed by baking at 80 degrees Celsius or UV ______.

Plasmid vectors are circular, ds DNA that is capable of independent replication. It is widely used in ________ techniques.

Phage based vectors are suitable for cloning larger fragments of DNA. Isopycnic CsCl gradient centrifugation is used to purify DNA molecules. The DNA sample is mixed with caesium chloride, CsCl, which forms a self-generating uniform density gradient during the centrifugation process. DNA molecules will sediment only to the position in the centrifuge tube at which the gradient density is equal to its own density, and they will remain there to form a band. After the DNA band has formed, it is removed from the centrifuge tube and the CsCl removed by precipitating the DNA with ________.

Genomic DNA may be further purified by using ________ charged matrices such as silica or DEAE in the presence of high salt that permits DNA binding but prevents the binding of contaminants.

Isopycnic centrifugation of DNA molecules includes the inclusion of ethidium bromide in the centrifugation mix, which results in its binding to DNA. The topological constraints of supercoiled DNA mean that closed-circular DNA molecules can be separated based on their ________.

DNA Ligase is an enzyme that is commonly used in molecular biology laboratories to join DNA strands together. It catalyzes the formation of a phosphodiester bond between the 3’-OH end of one DNA fragment and the 5’-phosphate end of another. This enzyme plays a crucial role in ________ DNA fragments.

DNA Polymerase I is an enzyme with 5’ to 3’ polymerase activity and 3’ to 5’ exonuclease activity. It is commonly used in DNA replication and repair processes. During replication, DNA Polymerase I is responsible for synthesizing a new DNA strand in the 5’ to 3’ direction. The exonuclease activity allows it to proofread and correct errors by removing nucleotides in the ________ direction.

PCR products lack a 5’-P, undergo ligation, separation of fragments on gel, denaturation with alkali, transfer immobilization on membrane, and incubation with labeled single stranded DNA or RNA as probe. Complementary sequences are detected on X-ray film. In combination, Phosphatase and Kinase may be used for ______________ probes.

Phosphatase is responsible for removing 5' phosphates from fragments of DNA prior to labeling with radioactive phosphate. Kinase is used for radiolabeling oligonucleotides, usually with 32P, for use as ______________ probes.

Sequence information is available to analyze from existing databases, design a suitable probe, screen a cDNA library, and identify a positive clone with 80% homology to ______________.

To raise antibodies specific to the protein of interest, peptide sequencing, functional homology, and an expression clone are used. If there is 80% homology to Pyruvate Kinase, then screen an expression library, design primers, amplify the gene, and identify a clone that expresses the protein of interest with sequence information from existing ______________.

Restriction fragments of DNA are prepared by using a restriction enzyme to create heavy weight fragments, which are then transferred onto nitrocellulose paper (blot) or gel sponge for further analysis. The normal β-globin allele, sickle-cell allele, and heterozygote allele can be distinguished through this method known as ______________.

In the process of Southern Blot, DNA fragments are cut by a restriction enzyme and then separated on a gel. Subsequently, the fragments are transferred onto a membrane and probed with a labeled single-stranded DNA or RNA. The resulting pattern on X-ray film helps in identifying specific alleles or genetic variations. This technique is fundamental for genetic analysis and molecular biology research, especially in the context of ______________ detection.