(13) Recombinant DNA Technology and Gene Cloning
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35 terms
Terms | Definitions |
|---|---|
 recombinant DNA | _________________ _____ refers to the joining of DNA molecules, usually from different biological sources, that are not found together in nature. |
| restriction enzymes, vector, host cell | recombinant DNA procedure: generating specific DNA fragments using _________ ________ -> joining these fragments with a _______ -> transferring the recombinant DNA molecule to a ______ ________ to produce many copies that can be recovered from the host cell |
| clones | The recovered copies of a recombinant DNA molecule are referred to as _________, and can be used to study the structure and orientation of the DNA. |
| restriction enzyme | A __________ ________ binds to DNA at a specific recognition sequence and cleaves the DNA to produce restriction fragments |
| palindromic | Most recognition sequences are _____________, and restriction enzymes often cleave these sequences in an offset manner. |
| DNA ligase | ____ _________ joins restriction fragments covalently to produce intact DNA molecules |
| vectors | ________ are carrier DNA molecules that can replicate cloned DNA fragments in a host cell. |
| vectors | ________ must be able to replicate independently and should have several restriction enzyme sites to allow insertion of a DNA fragment. |
| plasmid | A _________ is an extrachromosomal double-stranded DNA molecule that replicates autonomously in bacterial cells |
| restriction sites, selectable gene | Plasmids used for DNA cloning usually have been engineered to contain: a number of convenient ___________ _________; a __________ ____________ to select for its presence in the host cell |
| lambda phage | The central third of _________ _________ vectors can be replaced with foreign DNA without affecting the ability to infect cells and replicate. |
| polylinker region | allows a number of restriction enzymes to cleave but only once each so that plasmid will still be functional |
| cosmid | _________ vectors are created by combining parts of lambda phage and parts of plasmids. |
| lambda phage | bacteria virus used as vector, storage package for DNA that is going to be sequenced |
| lambda, plamids | Cosmids contain the cos sites of ________, which are necessary for packaging of phage DNA into phage particles. Once inside the bacterial cell, cosmids replicate as ___________. |
| Bacterial artificial chromosomes (BACs) | ___________ ___________ __________ are based on F factor and can carry up to 300 kb of inserted DNA |
| expression vectors | _________ ___________ are engineered to express a gene of interest to produce large quantities of the encoded protein. |
| yeast | __________ cells are used as eukaryotic hosts for cloning |
| Yeast artificial chromosomes (YACs) | ___________ __________ ___________ can contain 100-1000 kb of inserted DNA |
| microinjection | Transgenic mice can be produced by transferring YACs by __________ into the nucleus of a mouse oocyte |
| polymerase chain reaction (PCR), in vitro | The ____________ ________ ___________ copies a specific DNA sequence through __ _____ reactions that can amplify target DNA sequences present in very small quantities. |
| oligonucleotide primers | PCR requires two ____________ ____________, one complementary to the 3' end of one strand of the DNA to be amplified and one complementary to the 3' end of the other strand. |
| denaturation, annealing, extension | The three steps of PCR— __________, primer ____________, and _____________ are repeated over and over using a thermocycler to amplify the DNA exponentially. |
| genomic library | Ideally, a __________ __________ contains at least one copy of all the sequences in the genome of interest. |
| genomic library | complete collection of cloned DNA fragments from an organism |
| chromosome specific libraries | __________ __________ __________ are libraries made from subgenomic amount of DNA such as a single chromosome |
| cDNA | A _______ library contains complementary DNA copies made from the mRNAs present in a cell population and represents the genes that are transcriptionally active at the time the cells were collected for mRNA isolation. |
| reverse transcriptase | _________ ___________ PCR (RT-PCR) can be used to generate cDNA from mRNA |
| probes | ____________ complementary to part of a gene are used to screen a library to recover clones of a specific gene. |
| agar | To screen a plasmid library, clones from the library are grown on ______ plates to produce colonies. |
| filter, probe | The colonies are screened by transferring bacterial colonies from the plate to a _______ and hybridizing the filter with a nucleic acid ________ to the DNA sequence of interest |
| plaque hybridization | A phage library is screened by _________ ______________ |
| restriction map | A _________ ______ establishes the number and order of restriction sites and the distance between restriction sites on a cloned DNA segment |
| Southern blot | A __________ ______ is used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments from restriction digest. |
| dideoxy chain termination sequencing, Sanger | The most common method of DNA sequencing is ____________ _________ _________ ________, developed by ___________ |
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