Exam 2- molecular biology techniques Flashcards
Gene transfection
Inserting intact genes into cells in culture, which is usually followed by expression of genes in the introduced DNA. GFP can be linked to a gene of interest to monitor its function.
Gene editing
Adding/removing genes or single bases. This is accomplished by crispr-cas9.
How can gene expression be controlled?
Knockin and knockout transgenic animals and cells, siRNA, shRNA, and antisense RNA
Oxford Nanopore Technologies MinION
system
A portable device that sequences DNA and RNA in real time. Uses bacterial pores for nanopore sequencing. This is some of the technology that lead to the $1000 genome.
Transcriptome
The set of all RNA molecules in a cell or set of cells- think of DNA transcription.
Genome
Refers to all DNA molecules in a cell/set of cells
Proteome
All protein molecules in a cell/set of cells. Proteomics is the study of the entire proteome.
Epigenetics
Epigenetics are inheritable non base changes in DNA due to environment. It is modification of DNA and DNA associated proteins resulting in a change in gene function- these modifications include DNA methylation and histone phosphorylation
What is an example of an epigenetic change?
Cells in the bone marrow differentiate into either stem cells or progenitor cells- lymphoid and myeloid progenitor cells generate daughter cells that carry out different functions. Both types of progenitor cells have the same DNA sequence as the zygote they developed from, but their developmental potential is restricted due to the epigenetic differences between them.
What causes epigenetic changes?
The expression of transcription factors that regulate cell differentiation. They control the expression of other genes that encode transcription factors and proteins involved in cell to cell communication. The changes in gene expression due to transcription factors is maintained over multiple cell divisions by modification of histones and methylation of DNA.
Metabolome
Includes all other molecules- sugars, nucleotides, amino acids, and lipids. They are compounds for molecules that regulate the metabolism.
How does the metabolome relate to cancer?
Metastatic cancer cells spread in the body in a complex microenvironment. If we can better understand their decision-making process in this regard then
clinically attacking metastatic cancer cells may be easier. A group of researchers at Vanderbilt have shown that they pick the route that demands the least amount of energy which implies targeting the metabolome
may be important. They prefer large spaces rather than narrow passages, so they can be considered “lazy”.
Secretome
Analysis of secreted proteins, DNA, other molecules, and vesicles. These could serve as biomarkers for cancer, like cfDNA for example.
cfDNA
Cell free DNA- DNA shed from cancer cells and transported through the bloodstream, acts as a biomarker of cancer. Can be detected with appropriate instrumentation.
Importance of exosomes
Stem cells secrete factors in exosomes that are important to tissue repair and other physiological changes. Clinical trials now underway, and exosomes may replace stem cells in stem cell therapy in the future. Advantage- stem cells are forever, exosomes have a half life
Importance of exosomes in Covid
People with Covid can experience a cytokine storm- cytokines are released from the damaged tissue and cause an immune reaction.
Mesenchymal stem cell exosomes can be used as treatment. Stem cells are known to influence immunomodulation- change the functioning of the immune system.
How are mutations generated in cells?
Mutations are absolutely critical to cell and molecular biology. They can be caused by chemicals or UV light.
Temperature sensitive mutants
Single point mutation give rise to proteins that are unstable and non-functional at a slightly elevated temperature. Yeast mutants are an example- they cannot secrete proteins at a nonpermissive temperature
Secretory pathway
The secretory pathway carries proteins to the cell surface membrane where they can be released.
Permissive temperature
23 degrees C, at this temperature all temperature sensitive cells will grow
Nonpermissive temperature
36 degrees C, at this temperature, temperature sensitive mutants will die.
Why are yeast mutants important?
The secretory pathway and the components needed for vesicular trafficking are similar in all eukaryotic cells. Yeast cells can be used to study this pathway. When they are transferred from a permissive to a higher nonpermissive temperature, they accumulate secretory proteins at the point in the secretory pathway blocked by the mutation. Mutants can be studied based on their areas of protein accumulation and mechanisms of vesicular transport can be determined.
Benefits of yeast cells in studying mutations (4)
- Simple cell to grow
- Minimal cell culture medium
- Haploids and diploids
- Temperature sensitive mutants
Genetic complementation
Restoration of the wild type recessive phenotype is accomplished by mating two different mutants.
Complementation analysis
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
Purpose of complementation analysis
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.
What experiment is complementation analysis used for?
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
Why can’t complementation analysis be used for dominant mutations?
The phenotype produced by a dominant mutant allele is displayed even in the presence of wild type (natural) allele.
Genetic suppression
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.
Restriction nucleases
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.
Where and when were restriction nucleases discovered?
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
Restriction site
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.
Restriction fragments
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.
What are restriction nucleases used for?
Used in genetic identity (finger print) and forensic medicine. DNA fragments can also be inserted in vector DNA.
Blunt ends
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.
