Exam 2- molecular biology techniques Flashcards

Question

Complementation analysis

Answer 1

If two recessive mutations a and b are in the same gene, then a diploid organism will exhibit the mutant recessive phenotype since neither allele provides a functional copy of a gene. If mutations a and b are in a separate gene, it will result in heterozygotes with only one copy of the allele that will not exhibit the mutant phenotype. It is said that these alleles complement each other

Answer 2

A test to determine if two recessive mutations are in the same genes or different genes. To study cellular processes, researchers isolate recessive mutations that produce the same phenotype.

Answer 3

Used to distinguish individual genes in a set of functionally related genes that all function to produce a phenotypic trait. One example of a study that used this technique is a study examining how many genes were affected by cdc mutations in yeast

Answer 4

The phenotype produced by a dominant mutant allele is displayed even in the presence of wild type (natural) allele.

Answer 5

Genetic suppression occurs when the phenotypic defects caused by a mutation in a particular gene are rescued (suppressed) by a mutation in a second gene. If two proteins (A and B)'s normal function depends on the proteins interacting, a specific change in protein A could result in a compensatory change in protein B so the proteins could interact. If both suppressor mutations occurred, strains with both alleles would be normal. With only one allele, a mutant phenotype would be expressed.

Answer 6

Cleave DNA strands at restriction sites. They generate DNA fragments that have a single stranded "tail" at each end that are complementary to the tails on other fragments generated by the same enzyme. They are called sticky ends, and these fragments can base pair transiently.

Answer 7

They were discovered in bacteria in the 1970s- they received a Nobel prize in 1978. Prior to this, DNA was too long to analyze in a similar manner to proteins

Answer 8

Sequences of 4-8 base pairs. They are palindromic sequences, meaning that the reaction site sequence is the same when read in either direction on the DNA strand. Restriction enzymes cut DNA at this site, but they can also be blocked from cleaving the DNA by modification enzymes.

Answer 9

A restriction enzyme will cut DNA into a reproducible set of fragments called restriction fragments. The length of the recognition site determines the frequency with which the enzyme cuts DNA and therefore the size of the restriction fragments. The smaller the base pair sequence of the site, the more frequently the DNA is cut.

Answer 10

Used in genetic identity (finger print) and forensic medicine. DNA fragments can also be inserted in vector DNA.

Answer 11

Some restriction enzymes do not produce sticky ends (overhang) at the end of the DNA fragments. Blunt ends occur when an enzyme cuts straight down the middle of the DNA sequence.

Answer 12

Used in DNA replication and DNA cloning. If the ends of DNA fragments have matching (complementary) ends, DNA ligase can join them together and make an unbroken molecule. This can still work with blunt ends, but it's more difficult than it would be with sticky ends. This is because there are no overhangs to hold the molecules in place transiently, and more DNA ligase is required for blunt ends.

Answer 13

A restriction enzyme that comes from E. coli. It produces sticky ends

Answer 14

Circular, double stranded DNA molecules that replicate separately from a cell's chromosomal DNA. They are duplicated before every cell division and segregated to each daughter cell. They occur naturally in bacteria and in some eukaryotic cells such as yeast. Used in recombinant DNA technology

Answer 15

Using DNA ligase. Restriction enzymes are used to cut out the DNA of interest and to open up the vector. The restriction fragment DNA is added to the vector by mixing the two together in the presence of the enzyme DNA ligase

Answer 16

1. A replication origin (ORI) 2. A marker that allows selection of plasmids that have the incorporated gene (selectable marker), usually a drug resistance gene. 3. A region in which exogenous DNA fragments can be inserted.

Answer 17

Association of two complementary nucleic acid strands to form double stranded DNA molecules- can contain 2 DNA strands, 2 RNA strands, or a combination. Used experimentally to detect DNA or RNA sequences. The two strands associate (hybridize) with each other via base pairing.

Answer 18

1. FISH 2. Antisense RNA 3. Northern blots 4. Southern blots 5. cDNA microarrays 6. Molecular beacons

Answer 19

Researchers tried to inhibit the expression of a gene in C. elegans by injecting a single stranded complementary RNA that hybridizes to encoded (also single stranded) mRNA and prevents its translation.

Answer 20

Can be used to separate proteins and nucleic acids. The protein mixture is placed in gel and electrical current is applied. Smaller molecules can move more quickly through the gel, and spherical molecules move more quickly than asymmetric ones. Negatively charged molecules move toward the positive pole. Can separate polypeptide chains differing by as little as 10 percent in weight.

Answer 21

Gel/ Band Shift assays are designed to identify DNA binding proteins. Used to detect protein complexes with nucleic acids

Answer 22

Pathological cell death

Answer 23

Programmed cell death- this is how chemotherapy and radiation work

Answer 24

Used to separate DNA. Purified DNA from a biological sample is digested with a restriction enzyme(s), and the resulting DNA fragments are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.

Answer 25

The clinical analysis using VNTR’s to analyze bone marrow transplantation was accomplished using Southern Blots

Answer 26

Variable number tandem repeats. Since most human genes are the same from person to person, DNA typing relies on the stretches of DNA that tend to differ among different people. The repeated sequences themselves are usually the same from person to person, but the number of times they are repeated tends to vary. These stretches of repeats are called VNTRs and can be isolated from an individual’s DNA.

Answer 27

Scientists use polymorphic loci (regions on a chromosome) that are known to contain VNTRs in order to differentiate people based on their DNA. This is often used in forensic science or in maternity/paternity cases. a database called the Combined DNA Index System (CoDIS) that gathers data on a number of STRs. By establishing the number of repeats of a given locus, law enforcement officials can differentiate individuals based on the repeat length of these alleles.

Answer 28

Single nucleotide polymorphism (variation in one base). These polymorphisms may be the future of personalized medicine. They can be used to predict side effects and required doses of a drug for each individual. They could also be used to predict the severity of ASD. Some people might have a genetic variation predisposing them to more severe side effects or the drug not working.

Answer 29

Can determine both the molecular weight and relative abundance of mRNA in cells. It uses denaturing gel to separate RNA according to size, then the RNA is fixed to a membrane and a complementary probe is used to bind to the RNA of interest so it can be analyzed.

Answer 30

Non-radioactive method for detecting specific sequence of nucleic acid (DNA or RNA). Hairpin shape w/ internally quenched fluorophore- fluorescence restored when it binds to the target nucleic acid sequence. Hybridizes in situ more efficiently than FISH

Answer 31

Also called gene chips- another DNA microarray method. Multiple DNA oligonucleotides are synthesized from an initial oligonucleotide bound to a glass slide. Several oligonucleotide sequences corresponding to different regions of a single gene can be synthesized in neighboring regions of the slide. With this method, oligonucleotides representing thousands of genes can be produced on a single glass slide.

Answer 32

cDNA from mRNAs are fluorescently labeled. In a microarray, DNA spots representing genes that are expressed still hybridize to their complementary DNAs that can be detected using a scanning laser microscope

Answer 33

Used to examine changes in gene expression. cDNA was taken from fibroblasts grown in serum and labeled with different colored fluorescent dyes (green and red). The unhybridized cDNA is washed away, and the intensity of red and green fluorescence is used to measure the level of expression of that gene in response to serum. Genes that are hybridized at the same level in both conditions (serum and without serum) hybridize equally to both red and green labeled cDNA preparations.

Answer 34

Subtyping human cancer cell types

Answer 35

Cryosurgery of the prostate

Answer 36

They are reverse transcribed using reverse transcriptase

Answer 37

A refined RNA seq method where there are a few rounds of amplification of cDNA with PCR. This has increased the sensitivity of the method so that RNA abundance can be measured in single cells. It allows detection of differences in gene expression in individual cells that might be obscured if many different cell types were studied together.

Answer 38

Used to compare human stem cell cardiomyocytes to in vivo cardiomyocytes

Answer 39

Starts with a plasmid vector and the DNA fragment to be cloned. The DNA fragment is inserted into the plasmid vector using an enzyme. E. coli is mixed with the plasmids, and only the transformed cells survive. The plasmids will replicate and be passed down as the cells replicate

Answer 40

Starting material is all of the genomic DNA. Advantage- The gene of interest is present. Disadvantage- there is a huge library of E. coli DNA fragments to search

Answer 41

Starting material is mRNA not DNA. Advantage- smaller library to search. Disadvantages- need to convert mRNA to cDNA to clone E.coli using Reverse Transcriptase. Also, if the gene isn’t being expressed then the gene won’t be cloned

Answer 42

To create humulin, G-CSF cDNA and a lacZ gene is inserted into the plasmid vector. The plasmid vector is returned to E. coli bacteria. The recombinant bacteria then begins to produce humulin.

Answer 43

1. Typically don’t secrete proteins so difficult to harvest- protein present in inclusion bodies 2. Bacterial proteins are not post-translationally modified- glycosylation, phosphorylation, folding, etc. This is required for protein function

Answer 44

Experimental introduction of foreign DNA into culture, usually followed by expression of genes in the introduced DNA

Answer 45

1. Polymers 2. Optoperforation- laser assisted 3. Electroporation 4. Microinjection using microneedles 5. Lipid mediated 6. Viral transduction

Answer 46

1. Transient- short term 2. Stable transfection 3. Cells that are often used are CHO cells and HeLa cells

Answer 47

The plasmid vector enters a mammalian cell and replicates efficiently due to the replication origin. Many plasmids are generated for the protein to be expressed from. However, plasmids are sometimes not segregated into daughter cells and therefore a large amount of cells in culture will eventually not contain a plasmid

Answer 48

Integrates the vector into the genome of the host cell. The genome is permanently altered . G-418 is a compound used to kill the cells without the desired gene- only the cells that have integrated the expression vector into the host chromosome will survive and clone themselves

Answer 49

A biologic for rheumatoid arthritis. TNF is a naturally occurring cytokine that is involved in normal inflammatory and immune responses. It plays an important role in the inflammatory processes of RA. ENBREL was designed to bind to TNF and block its interaction with cell surface TNF receptors

Answer 50

Polymerase chain reaction. DNA is denatured (heated) into single strands and oligonucleotides are added that are complementary to the target sequence- they hybridize to the DNA. The area of the DNA bound by oligonucleotides act as primers for DNA chain synthesis as temperature cools. After multiple cycles, the DNA sequence is amplified,

Answer 51

Using transcription to go from RNA to DNA. Uses reverse transcriptase. This is a function of retroviruses.

Answer 52

A temperature resistant DNA polymerase, can remain active even when heated to 95 C. Used in PCR

Answer 53

Jackson Laboratory – animals made specifically for the addition (transfected gene) of “knockout” of a gene for diabetes, Parkinson’s, etc.

Answer 54

A mouse that is deficient in its ability to express a selected gene of interest. If the mouse's cells can't produce telomerase associated RNA, their telomeres will shorten excessively until they can't reproduce

Answer 55

Add and create a mutation in a gene.

Answer 56

Short stranded RNA that base pairs with specific target mRNAs, causing their degradation and preventing gene expression. RNA interference is an important defense against viral infection

Answer 57

1. Simple methodologies 2. Fast and efficient transfection 3. Modifications are available

Answer 58

1. Non renewable 2. Knockout is only transient 3. siRNA can't be easily transfected into all cell lines

Answer 59

A double stranded form of RNA. To produce in vivo, a synthetic gene is expressed, containing segments of sense and antisense sequences corresponding to the target gene. Once it's produced, shRNA can be cleaved to produce siRNA

Answer 60

1. Renewable resource 2. Transient or stable knockdown 3. Transfection or viral delivery 4. Viral delivery to most cells, even hard to transfect cells

Answer 61

1. Technically challenging 2. Design rules are less understood

Answer 62

No cure for Huntington’s Disease – causes breakdown of nerve cells in the brain. HD is due to an overabundance of huntingtin which, in the form of siRNA, is toxic to nerve cells but especially to cancer cells. HD patients have 80% fewer cases of cancer. This information could potentially be used to develop a new cancer treatment. Recent study shows that siRNA tethered to nanoparticles reduced human tumor growth in mice w/o toxicity

Answer 63

Clustered Regularly Interspaced Short Palindromic Repeats. Researchers create a small piece of RNA with a short "guide" sequence that attaches (binds) to a specific target sequence in a cell's DNA, much like the RNA segments bacteria produce from the CRISPR array. This guide RNA also attaches to the Cas9 enzyme. When introduced into cells, the guide RNA recognizes the intended DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location, mirroring the process in bacteria. Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material

Answer 64

Antisense is the non-coding DNA strand of a gene. In a cell, antisense DNA serves as the template for producing messenger RNA (mRNA), which directs the synthesis of a protein

Answer 65

hESCs – Human Embryonic Stem Cells – feeder layers used in cell culture are most commonly used when growing these cells. hESCs are NOT derived from the 4 stage human embryo. Disease states possibly treated/cured with stem cell therapy: Parkinson’s disease, Duchenne’s muscular dystrophy, blindness