| what are restriction endonuclease? | An enzyme that recognizes specific short sequences of DNA and cleaves the duplex (sometimes at the target site, sometimes elsewhere, depending on type). |
| what are nucleases and phosphatases? | Nucleases hydrolyze an ester bond within a phosphodiester bond,Phosphatases hydrolyze the ester bond in a phosphomonoester bond. |
| what is the difference between endonuclease and exonuclease? | endonuclease – Nuclease that cleaves phosphoester bonds within a nucleic acid chain (It may be specific for RNA or for single-stranded or double-stranded DNA).
exonuclease – Nuclease that cleaves phosphoester bonds one at a time from the end of a polynucleotide chain (It may be specific for either the 5′ or 3′ end of DNA or RNA). |
| what can restriction endonuclease be used for? | it can be used to cleave DNA into defined fragments. |
| what can be generated using the overlaps of fragments? | A map can be generated by using the overlaps between the fragments generated by different restriction enzymes. |
| what are cloning vectors? | DNA ( derived from a plasmid, bacteriophage genome) that can be used to propagate an incorporated DNA sequence in a host cell.Vectors contain selectable markers and replication origins to allow identification and maintenance of the vector in the host. |
| what cloning a DNA fragment require ? | a fragment of DNA requires a specially engineered vector. |
| what is recombinant DNA ? | A DNA molecule that has been created by joining together two or more molecules from different sources. |
| what does subclone mean? | The process of breaking a cloned fragment into smaller fragments for further cloning. |
| what is a MCS? | Multiple cloning site is a sequence of DNA containing a series of tandem restriction endonuclease sites used in cloning vectors for creating recombinant molecules. |
| what are cloning vectors divided into? | Can Be Specialized for Different Purposes. |
| how can DNA be introduced into different target cells? | with these methods |
| what's a probe? | A radioactive nucleic acid, DNA or RNA, used to identify a complementary fragment. |
| what's autoradiography (nucleic acid detection) | A method of capturing an image of radioactive materials on film. |
| what's in situ hybridization? | Hybridization of a probe to intact tissue to locate its complementary strand by autoradiography. |
| what are the DNA sequencing steps? | Gel electrophoresis separates DNA fragments by size, using an electric current to cause the DNA to migrate toward a positive charge. |
| what is the PCR and RT-PCR tesuniqes? | Polymerase chain reaction permits the exponential amplification of a desired sequence, using primers that anneal to the sequence of interest,RT-PCR uses reverse transcriptase to convert RNA to DNA for use in a PCR reaction. |
| what is southern blotting ? | involves the transfer of DNA from a gel to a membrane, followed by detection of specific sequences by hybridization with a labeled probe. |
| what is northern blotting ? | is similar to Southern blotting, but involves the transfer of RNA from a gel to a membrane. |
| what is western blotting? | entails separation of proteins on a sodium dodecyl sulfate (SDS) gel, transfer to a nitrocellulose membrane, and detection of proteins of interest using antibodies. |
| what's an epitope tag? | A short peptide sequence that encodes a recognition site (“epitope”) for an antibody, typically fused to a protein of interest for detection or purification by the antibody. |
| what are microarrays? | they comprise known DNA sequences spotted or synthesized on a small chip. |
| what's next generation sequencing (NGS), what does it refer to? | Next-generation sequencing is based on the ability to sequence, in parallel, millions of DNA fragments,
Next generation sequencing (NGS) refers to large-scale DNA sequencing technology that allows for querying
- the entire genome (whole genome),
- the exons within all known genes (whole exome),
- or only exons of selected genes (target panel). |
| how is genome-wide transcription analysis is performed? | is performed using labeled cDNA from experimental samples hybridized to a microarray containing sequences from all ORFs of the organism being used. |
| what are SNP arrays? | they permit genome-wide genotyping of single-nucleotide polymorphisms. |
| what is RNA sequencing? | uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment. |
| what does transgenics mean? | Organisms created by introducing DNA prepared in test tubes into the germline.
The DNA may be inserted into the genome or exist in an extrachromosomal structure. |
| Embryonic stem (ES)? what do they contribute to? | cells that are injected into a mouse blastocyst generate descendant cells that become part of a chimeric adult mouse.
-When the ES cells contribute to the germline, the next generation of mice may be derived from the ES cell.
-Genes can be added to the mouse germline by transfecting them into ES cells before the cells are added to the blastocyst. |
| how can an endogenous gene be replaced? | be replaced by a transfected gene using homologous recombination. |
| how can a successful homologous recombination be detected? | by using two selectable markers, one of which is incorporated with the integrated gene, the other of which is lost when recombination occurs. |
| what are knockouts and knock-ins in the cre//ox ? | knockout – A process in which a gene function is eliminated, usually by replacing most of the coding sequence with a selectable marker in vitro and transferring the altered gene to the genome by homologous recombination.
knock-in – A process similar to a knockout, but more subtle mutations are made. |
| Array comparative genome hybridization? | (array-CGH) allows the detection of copy number changes in any DNA sequence compared between two samples. |
| what has the Introduction of NGS has resulted in ? | -a dramatic increase in speed and content of sequencing
-at a fraction of the cost |
