Term
| What is the benefit of cloning genes? |
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Definition
Gene cloning- isolation of a specific DNA sequence in a way that makes it possible to manipulate it in the lab
- In order to fully understand molecular basis of many processes, traits, or diseases, it is very helpful to have DNA sequence info of key genes |
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Term
| Targeted cloning approach |
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Definition
| solation of specific genes that we already know exist based on mapping studies or presence of mutants |
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Term
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Definition
| Isolation of many genes that may have something to do with a process of interest, without a prior knowledge of existence and/or function of genes; generally lies on induction of mutations followed by a screen for altered phenotypes |
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Term
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Definition
| A novel DNA sequence formed by the combination of two nonhomologous DNA molecules |
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Term
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Definition
| a carrier of recombinant DNA, and is normally a plasmid or viral DNA origin |
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Term
| How do you create a vector? |
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Definition
| use same restriction enzyme to cut plasmid DNA that was used to develop DNA fragments that are inserted into the plasmid |
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Term
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Definition
- PCR: in vitro amplification of DNA
- Developed by Dr. Kari Mullis in 1983 (got Nobel prize in chemistry)
- Relies of DNA polymerases from thermophilic archaebacteria (can survive a very hot temp)
- Requires primers- oligonucleotides of 18-25 nucleotides long |
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Term
| What are the 3 steps of PCR? |
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Definition
o 1. Denaturation- melting two DNA strands by heating to 94 C
o 2. Primer annealing- cool reaction to temp between 50 and 70 C
Primers have known sequence and delineate the region that will be amplified
Primers are designed by researcher and synthesized by companies
o 3. Primer extension- at 72 C using heat-stable polymerase
o Happens in machine called thermal cycler
o Amplification is exponential |
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Term
| What is the original Sanger sequencing? |
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Definition
sequencing reactions were carried out in four separate tubes
Incorporation of ddNTP blocks further synthesis
Each reaction loaded in a separate lane; products separated on a gel; sort fragments at bottom
Gel is dried, exposed to X-ray film, bands read from bottom to top |
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Term
| What is the modern Sanger sequencing? |
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Definition
Each ddNTP has its own fluorescent label
All four ddNTPs are included in sequencing reaction
Products separated on polyacrylamide gel, but more commonly using a thin capillary tube
A laser at bottom of gel or tube scan which label passes |
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Term
| Map-based cloning/sequencing |
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Definition
o Relies on fragmentation of chromosomes into fragments of 150 kb in length that are cloned in bacterial artificial chromosomes
o A shortest tiling path is constructed from these BACs using molecular markers
o BACs then fragmented and pieces are cloned, sequenced, and aligned to BAC
o Used in Human Genome Project |
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Term
| Whole-genome shotgun cloning/sequencing |
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Definition
o Entire genome sheared in small, overlapping pieces 3-5 kb in length, which are cloned into plasmids and sequenced
o Overlapping sequences are aligned with use of computer programs and assembled to represent the entire genome
o Privately funded Celera Genomics used this and it was faster |
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Term
| Natural process of agrobacterium |
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Definition
| bacteria injects plasmid into plant cell DNA |
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Term
| Engineered process of agrobacterium |
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Definition
Foreign DNA + shuttle vector = plasmid
‘Binary system’ within bacteria is bacterial chromosome, shuttle vector plasmid, and TI plasmid
Shuttle vector plasmid is injected and incorporated into plant DNA |
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Term
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Definition
o Agrobacterium is soil-borne pathogenic bacterium that induces crown gall in susceptible plants
Involves transfer of DNA from tumor inducing (TI) plasmid of bacteria to plant
Plant tumor makes compound bacterium needs (opines, nopalines)
TI-genes can be removed and replaced by gene of interest
TI DNA can be moved into second plasmid |
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Term
Gel electrophoresis:
-amount of DNA used
-what is gel made of
-what is gel submersed in
-direction of DNA |
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Definition
o Amount of DNA is between 10 nanogram and 20 microgram
o Gel made of agarose (polysaccharide from seaweed) that gels after boiling
o Gel submersed in low-salt buffer to allow electricity to go through, maintain pH
o DNA is neg charged from phosphate groups, so DNA migrates to positive end
o Gel can be used to visualize DNA, estimate size based on a size marker of known fragments; DNA can be isolated from the gel if desired |
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Term
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Definition
o Cloned DNA segment is small and may represent only single gene or portion of gene
o Many clones needed to cover organism’s genome
o Set of DNA clones is called a cloned library |
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Term
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Definition
o DNA strand synthesized from mature mRNA
o Reverse transcriptase is enzyme responsible for synthesizing
o Contains uninterrupted sequence of eukaryotic gene
No introns
Can still function to express desired gene product |
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Term
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Definition
organisms that do not carry a correct set of their basic chromosome number
o Organisms gains or loses one or more chromosomes, but not complete set |
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Term
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Definition
| the loss of a single chromosome |
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Term
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Definition
| the gain of a single chromosome (3 homologous chromosomes) |
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Term
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Definition
| an individual having more than 2 sets of the basic chromosome number |
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Term
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Definition
(x)
o Lowest chromosome number in polyploid series
o Each chromosome represented only once
o Haploid from diploid species contains basic chromosome number |
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Term
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Definition
| multiple sets of chromosomes are identical |
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Term
| formation of autopolyploid |
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Definition
• 1. Failure of chromosomes to segregate during meiotic divisions can produce diploid gamete
o Fertilization by a haploid gamete triploid zygote
• 2. Crossing a diploid with a tetraploid
o Diploid gamete: x chromosmes
o Tetraploid gamete: + 2x chromosomes
o Triploid zygote: 3x chromosomes |
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Term
| why are triploids sterile and why are they valuable? |
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Definition
| - Odd number of chromosomes fail to divide evenly and create unbalanced gametes |
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Term
| How do restriction enzymes work? |
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Definition
| - They locate the restriction site on the gene but then they cut it as a sticky end of blunt end |
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Term
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Definition
X-Gal converted by beta-galactosidase into Gal and X (X by itself is blue)
Bacteria without plasmid die- no colony
Bacteria with empty plasmids are blue because they have functional lacZ
Bacteria with recombinant DNA are white because lacZ has been interrupted |
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Term
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Definition
o DNA can be shot into cells- known as biolistics or particle bombardment
o DNA precipitated onto small gold or tungsten particles
o Gene of interest under control of a suitable promoter
o Selectable marker (antibiotic/herbicide resistant gene) to identify cells that stably express the transgene
o Particles shot into cell using pressure burst
o Percentage of cells that take up DNA > % that express transgene > % that stably express transgene
o Hard to control copy number- some transformed cells have many copies, others few
o Agrobacterium transformation results in low number of transgenes and more predictable results
o Also important from regulatory perspective |
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Term
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Definition
| multiple sets of chromosomes are different |
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Term
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Definition
| loss of chromosomal regions due to breaks |
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Term
| what are the 2 types of deletions |
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Definition
o Terminal
Deletion occurs near end of chromosome
o Intercalary
Deletion occurs within interior of chromosome |
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Term
| what are deletions dependent upon |
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Definition
- Loss depends on centromere
o Retained if broken portion contains centromere
o Segment without centromere is lost |
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Term
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Definition
| normal homolog must develop a bundle (compensation loop) to pair with chromosome that has deletion |
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Term
| what are consequences of deletions |
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Definition
o Occurrence in non coding are silent
o Single gene affects or large deletions that KO several closely linked genes are often lethal |
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Term
| duplications (insertions) |
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Definition
| - Increased copies of specific chromosome region (a region is present twice) |
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Term
| what are the causes of insertions |
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Definition
Unequal crossing over between synapsed chromosomes during meiosis
Duplication and deletion are created
o 2. Replication error prior to meiosis |
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Term
| what are consequences of insertions |
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Definition
| - Most duplications have no obvious phenotypic consequences and can only be detected by cytological or molecular methods |
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Term
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Definition
- Chromosome segment turned around 180 degrees within a chromosome (ABCDEF ABEDCF)
- Not a loss of genetic info
- Rearrangement of liner gene sequence
o Orientation of genes in segment is reversed
- Requires two break and subsequent inversion of inverted segment |
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Term
| what are the 2 types of inversions |
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Definition
o Paracentric
Inversion with ONE ARM of a chromosome
o Pericentric
Centromere is part of inverted segment. BOTH arms involved |
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Term
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Definition
| - Movement of a chromosomal segment to a new location in the genome |
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Term
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Definition
| o Exchange of chromosomal regions between two non-homologous chromosomes |
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Term
| what is the result of a translocation |
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Definition
| - Typically do not result in a loss of genetic material so they often do not cause detectable changes |
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Term
| robertsonian translocation |
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Definition
| o Non reciprocal translocation in which two acrocentric nonhomologous chromosomes fuse to form a single chromosome |
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Term
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Definition
| position that a gene occupies in a chromosome (gene location) |
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Term
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Definition
| alternative forms of a gene; found at same place on chromosome |
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Term
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Definition
| appearance of an organism |
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Term
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Definition
| genetic constitution of an organism |
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Term
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Definition
| crossing parental varieties that differ in only one trait of interest |
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Term
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Definition
| parents differ for two traits of interest |
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Term
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Definition
| parents differ for three traits for interest |
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Term
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Definition
| inherited different alleles at one or more loci; having different alleles of a gene between homologous chromosomes |
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Term
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Definition
| inherited identical alleles at one or more loci; having the same allele of a gene in homologous chromosomes |
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Term
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Definition
| a set of one maternal chromosome and one paternal chromosome that pair up with each other inside a cell during meiosis |
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Term
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Definition
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Term
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Definition
| all other cells besides sex (value = 2n) |
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Term
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Definition
| sometimes also called a pure-bred, is an organism that always passes down a certain phenotypic trait to its offspring |
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Term
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Definition
| controlled by single gene, not altered by enviro |
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Term
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Definition
| cross between dominant phenotype and a recessive phenotype; used to determine the genotype of the dominant plant |
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Term
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Definition
| the phenotypic effect of one allele completely masks the other in heterozygous combination; that is, the phenotype produced by the two alleles in heterozygous combination is identical to that produced by one of the two homozygous genotypes |
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Term
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Definition
| asexual reproduction in plants, in particular agamospermy |
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Term
| Mendel's Law of Segregation |
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Definition
| o During gamete formation, paired alleles of a gene segregate randomly so that each gamete receives one allele or another with equal probabilities |
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Term
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Definition
o Traits are determined by genes
o Gene exists in pairs of alleles |
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Term
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Definition
| o Results in uniform F1 progeny controlled by expression of the dominant allele |
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Term
| Mendel's Law of Independent Assortment |
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Definition
| o During gamete formation, segregation of alleles at one locus in INDEPENDENT of the segregation of alleles at another locus |
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Term
| How is recombinant DNA made using restriction enzymes |
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Definition
| - Restriction enzymes are used to produce recombinant DNA because they are sequence specific and they are able to recognize and be bound to specific DNA sequences. When mixed together, the molecules can join together through the base pairing on their sticky ends and the bond is made permanent by a DNA ligase which forms covalent bonds on the backbone of each DNA strand |
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Term
| What are the 3 properties of a vector? |
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Definition
o 1. Vector can be introduced into host cell
o 2. Vector contains replication origin and can replicate inside host cell
o 3. Cells containing vector can usually be selected by straightforward assay by allowing growth of host cell on selective media |
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