Unit 12: Molecular biology techniques Flashcards
define locus
specific site of a gene on a chromosome
define allele
one of two or more alternative forms of a gene
Define polymorphism
Presence of more than one allele of a gene/locus in a population at a frequency greater than that of new arising mutation
genotype vs. phenotype
genotype: entire genetic makeup
phenotype: physical and physiological characteristics. the specific trait associated with a particular allele
describe dominant mutations
- when the mutant phenotype is expressed with the WT allele present
- typically gain of function that results in increased activity and new function
- there is sometimes loss of function
name and describe types of loss of function in dominant mutations
haploinsufficiency: one gene copy is inactive, the functional copy does not provide enough gene product for the WT phenotype
dominant-negative: mutation interferes with WT protein function
describe recessive mutations
- WT phenotype expressed with WT allele present
- typically gene is inactivated partially or completely resulting in loss of function
Loss of function mutations vs. conditional loss-of-function mutation
- these are null mutations
- loss of function mutations include point mutations and deletions. these completely get rid of gene function
- conditional loss-of-function mutations depend on temperature. Usually dysfunctional at high temperatures
Describe conditional mutations
- mutations cause proteins to be functional only under certain conditions such as temperature
- permissive temperatures produce the WT phenotype
- non permissive temperatures produce the mutant phenotype
- this helps us study lethal mutations
what is the purpose of genetic complementation experiments?
genetic complementation- growth indicates different loci b/c of normal function
non-complementation- no growth indicates same loci but different alleles
what is the purpose of epistasis experiments?
experiments help determine order of function of gene products in a pathway
explain how gene loci are mapped in the genome
- gene loci are mapped by determining distance to known loci
what is complementation
- when several genes contribute to phenotype
- crossing heterozygotes with mutant recessive alleles in different loci will restore WT phenotype
what is epistasis
one gene’s alleles mask the effect of another gene’s alleles
what are single nucleotide polymorphisms (SNP)
single base sequence variation between individuals at a particular point in the genome
what are the required components and general process of DNA cloning
- generate recombinant vector
- transfer recombinant vector into host cell
- produce multiple copies of recombinant DNA by host
- select/identify recombinant host cells
how do you generate a recombinant vector
- cut source DNA at edges of gene
- select a suitable carrier DNA (vector) that is capable of self replication in a host
- insert gene covalently into vector
explain the principle of electrophoresis and how it is applied to DNA
separate based on mass using electric field
- it is used for
1. qualitative DNA analysis (determine purity of plasmid prep)
2. DNA purification: isolation of restriction fragments from gel
3. DNA-protein interaction studies: electrophoretic mobility shift assay
what do restriction enzymes do?
catalyze single strand breaks (nicks) on either DNA strand or double-strand breaks in DNA
- they hydrolyze phosphodiester bonds of DNA. producing free 3’-OH group and 5’-phosphate group
how is host DNA protected from restriction enzymes
methylation
where do restriction enzymes cleave on foreign DNA
restriction enzyme cleaves unmethylated foreign DNA within 4-8 base pair recognition site
- they recognize inverted repeats in DNA (aka palindromes) and cleave at the 3’ or 5’ site of/or at symmetry axis
are restriction enzymes used to cut out DNA segments of interest?
yes
define palindrome
- inverted sequence repeats that read in the same direction on each strand
what does choosing a cloning vector depend on?
- size of DNA insert
- efficiency of the host cell to uptake vector
- amplification
the three essential properties cloning vectors must possess
- restriction site that allows insertion of target DNA
- autonomous replication in a host cell so that amplification of target DNA can occur independent of host DNA synthesis
- a selectable feature that allows transfected host cells containing the vector to be identified from other cells w/o vector
describe plasmid vectors
- circular molecules
- replicate autonomously
- transfer to host via transformation
- selective marker is antibiotic resistance gene(s)
- can accommodate up to 15 kb
describe bacteriophages
- viruses infecting bacteria (e.g. lambda)
- transfer to host via transduction (aka phage infection)
- can insert up to 15-25 kb
describe cosmids
- plasmids with lambda segments or lambda phages
- accommodate larger inserts
- can hold up 20 kb
describe bacterial artificial chromosomes (bac)
- plasmids for very large fragments
- can hold 150-350 kb
- maintained at low copy numbers due to par genes regulating distribution of plasmids at division
- includes selectable markers and genes that regulate plasmid distribution
Yeast artificial chromosomes (YAC)
- contain all elements required for maintenance in eukaryotic cells ( origin, centromere, telomeres) and selective markers
- Circular and linear form
circular for rapid replication and stable storage
removable product that linearizes for transformation and restriction site for cloning - can insert 150-3000 kb
what are DNA libraries
collection of DNA molecules stored in vectors
genomic libraries vs. cDNA libraries
Genomic libraries:
- store complete genome of an organism
- tissue- independent
- contain complete coding region, including introns
- obtained from restriction enzyme digestion of the entire genome of an organism
- vectors used: plasmids, cosmid, lambda phage, BAC, YAC
- large fragments
cDNA (complementary DNA) libraries:
- store mRNA converted to DNA of a cell/tissue.
i.e all expressed genes of particular cell/tissue
- obtained from the mixture of mRNAs expressed by a particular tissue under given physiologic conditions
- clones lacks introns and a promoter region
enzymes: spliceosome, reverse transcriptase, and DNA polymerase
- vectors used: plasmid, lambda phage,
- small fragments
methods that use nucleic acid hybridization
- Gene analysis- based on reversible denaturation of DNA and complementarity
- southern blotting
- northern blotting
- western blotting
- eastern blotting
- south-western blot
what does gene analysis by hybridization utilize
probes- short ssDNA or RNA molecules complementary to specific region. are meant to hybridize with target DNA
- they are labeled for detection using radioactive isotopes, fluorescent/biotin tags
- their design is based on sequence formation
describe southern blotting
- DNA is denatured during transfer onto filter membrane. this allows probes to access.
- uses labeled DNA probes
- filter hybridized with probe to detect specific DNA fragment
- detects presence or absence of genes or alleles
- detects specific restriction fragment out of the millions present in a restriction digest
describe northern blotting
- detects RNA with labeled DNA probes
- useful in determining whether a specific mRNA is expressed in a particular tissue
describe western blotting
- detects proteins by using labeled antibodies
describe south-western blotting
-detects DNA-binding proteins with labeled DNA probes
- useful for detecting expression of transcription factors that interact with regulatory sequences controlling specific genes
describe eastern blot
- detects posttranslational modifications
what does PCR do?
amplifies specific regions of DNA in vitro replication using DNA polymerase
-oligonucleotides act as replication primers
- forward and reverse primers selected to flank target region
- does not require cloning
steps of PCR
- denature template using heat
- lower temp to allow hybridization of primers
- extend strand using heat-stable DNA polymerase
- repeat multiple cycles (25-45)
heat stable taq polymerase
what are PCR applications
- exponential amplification of DNA: increases efficiency in recombinant DNA procedures
- isolation and subcloning of specific segment of genomic DNA
- design of primers with restriction site to facilitate sub cloning - preparation of DNA probes
- diagnostics
- quantification of DNA or cDNA by qPCR (quantitative/real time PCR)
- forensic DNA analysis
- Gene expression analysis by Reverse transcriptase PCR
what is the Sanger dideoxy method
- in vitro synthesis of one strand (template based)
- uses didoexynucleotides to interrupt DNA synthesis using the unknown DNA as the template
- length is determined by the last base in sequence
describe Sanger dye termination sequencing
- daughter strand is denatured and separated by electrophoresis
- capillary gel electrophoresis separation for high resolution
- automated process using sequnators
how is the efficiency of net-generation sequencing achieved?
through pre-amplification of templates by PCR
how are genome sequences accessible
through databases:
- Genbank
- EMBL
How are sequences compared
using BLAST (Basic local alignment search tool)
- nucleic acid or protein sequences
- uses similarity scores (p-values)
ways of whole genome sequencing
early on: physical mapping and ordered sequencing of restriction fragments
shotgun approach: random sequencing and in silico assembly of single contiguous assemblies
gene expression studies
- northern blotting- mRNA expression. through electrophoresis separation, denaturation and blot transfer, hybridization w/ mRNA specific probe for mRNA detection
- semi-quantitative comparison w/ standard mRNA signal - in situ hybridization- detection of mRNA using labeled DNA or RNA probes. analysis of spatial and/or temporal expression patterns
- DNA microarrays- uses high density DNA chips to screen expression of large # of genes
- isolates total mRNA, requires RT, and flourescently labeled dye & hybridization
- allows analysis of comparative expression in clinical and lab settings
gene functional studies
- in vivo gene expression
- genetically modified animals
novel methods for gene expression analysis
- RNAseq- applying sequencing methods to RNA for transcriptome analysis. does not require prior knowledge of RNA sequences
- ribosome profiling- identify mRNA being actively translated
-isolated RNA and RNAse digest of free RNA followed by sequencing
-isolation and sequencing of remaining RNA
- requires reverse transcription of RNA sequences into cDNA
strategies to manipulate gene expression
- in vivo gene expression- transfection of foreign DNA into eukaryotic cells
- constitutive (continuous) or inducible (conditional) expression
- untagged or tagged target protein
- types of transfection: transient- expression is from cDNA in plasmid DNA; Stable (transformation)- expression from cDNA is integrated into host chromosome - genetically modified animals-
transgenic animals: animals carry foreign genes into genome and pass to offspring.
-by injecting trans gene into fertilized egg, random insertion by NHEJ, Selection and breed offspring
knockout animals: targeted gene inactivation by HR (deleted gene)
knockin animals: targeted gene replacement (modified gene) - IoxP Cre recombination system- for tissue specific targeted gene disruption by somatic cell recombination.
-viral recombinase Cre recognizes specific IoxP sequences. cleaves intervening sequences between IoxP sites.
- targeted gene knockout - CRISPR- bacterial defense using non-coding RNA
- targets bacteriophages.
integrates phage DNA fragment into specific genomic regions
when reinfected, sequences cleaves due to complementarity w/non-coding RNA
