For Exam 3 Flashcards
Forward Genetics
Start with a phenotype and identify the underlying gene(s)
Reverse Genetics
Stat with the DNA sequence of a gene and identify the encoded phenotype(function)
Uses the genetic code to predict the amino acid sequence of the protein, computational methods to predict protein structure and function, experimental methods to mutate the gene and see the effect on the organism.
DNA sequencing
Common methods of DNA sequencing rely on measuring the sequential incorporation of bases as DNA is synthesized.
The Sanger method used chemically modified nucleosides (ddNTPs) in combination with normal nucleosides (dNTPs).
Synthesis stops when a ddNTP is added by DNA polymerase to a polynucleotide chain because ddNTP has no hydroxyl group at the 3’ position.
The sequence is determined by detecting the terminal (3’) base (A,C,G,or T) at each position in the DNA
What ratio is ddNTPs to dNTPs?
DNTPs is higher rate/ratio then ddNTPs
Components of sequencing reactions:
-dna polymerase
-short(18-22 bases) primer complementary to template stand
-all 2’ deoxynucleoside triphophastes (dATP, dCTP, dGTP, dTTP)
-all 2’, 3’ dideoxynuclosides triphospates (ddNTPs); labeled uniquely so that each can be detected
-template DNA, sequencing reaction reliably read only 100-700 bases of a template
*ration is important
High-throughput sequencing
Used to simultaneously sequence many different template molecules
They involve physical binding of template DNA to a solid surface or to microbeads and amplification of DNA templates by PCR
Thousands or millions of sequencing reactions are run at once: massively parallel DNA sequencing
Sequencing assembly
-sequencing methods produce limited read sizes
-the key to sequencing a genome sequencing is to generate many short sequence fragments
-if a random method is used to generate the fragments, the fragments overlap
-the fragments can be arranged identifying regions of overlapping sequence using computer bases computational methods (genome assembly)
Assembling reads into chromosome sequence
Many individual short reads can be assembled into a longer contig of DNA sequence by orgaizing the overlapping bases
Funcational genomics:
Identify and annotate funcation to various parts of the genome
Open reading frames:
DNA sequence that contains codons for amino acids with no stop codons and thus may encode parts of proteins. Function of the protein can be predicted using computational methods or mutations
Comparative genomics
Compare genomes both writhing and across species
Addresses question: what genes are present in all species, how rapidly nucleotide and amino acid
RNA sequencing
Transcriptome :Subset of the genome that is expressed as RNA in a particular cell or tissue at a particular time
-determination of which genes are expressed in what amount can made by RNA sequencing (RNA-seq)
-DNA copies of the RNAs must first be synthesized using reverse transcriptase from RNA viruses
Protein analysis
Proteomics: study of proteome- the complete complement of proteins produced by an organism
Many genes encode more than one protein; alternative splicing and Posttranslational modifications increase the diversity of proteins derived from one gene.
The proteome can be measured using gel electrophoresis and mass spectrometry or using antibodies
Functional genomics
2d tell elctrophoresis
Mixture of extracted proteins seperated initially based on ionic charge considering different amino acid sequences vary
Secondarily, proteins separated based on mass
Metabolome
Metabolomics: comprehensive analysis of metabolites in a biological specimen
Metabolome: the complete set of small molecules in a cell, tissue, or organism
-primary metabolites: involved in normal cell processes; includes hormones and other signaling molecules
-secondary metabolites- often unique to particular organisms; antibiotics and chemicals made by plants for defense
Prokaryotic genomes
Key features of prokaryote genomes:
Relatively small, usuall one circular chromosome
Compact- mostly protein-coding regions and RNA genes
-most genes do not have introns
Often carry plasmids-small circular DNA molecules
*great diversity amount prokaryote genomes=huge variety of environments they occupy
Comparative genomics
Patterns:
-# of genes shared by all prokaryotes is small(implies only a few hundred genes required for life)
-core genome: containing genes common to all strains of a species
-pan genome is all genes found in a species across all strains
Minimal genome
Mycoplasma genitalium (only 483 protein coding genes)
Gene families
Genes are often present in many copies in eukaryotes as a result of duplication events.
Following duplication, the original and the copy can be mutated. If the gene product is nonfunctional, the gene is called a pseudogene or a mutated copy may be beneficial (on going evolution)
Paralogs: genes that arise via duplication. Two or more paralogs in a genome make up a group of closely related genes called a gene family.
Globin gene family
Different members of the gene family are paralogs, because they have arisen through gene duplication.
During human development, different globin genes are expressed at different times and in different tissues.
Non-protein coding DNA
In multicellular eukaryotes less than half of the genome encodes proteins. The rest includes introns, regulatory sequences, RNA genes, pseudogenes, and intergenic (non gene related) regions
The intergenic regions are mostly composed of repetitive DNA sequences including genome parasites such as a transposable elements.
Intergenic regions vs. Genes and gene related sequences
Intergenic regions make up about 60% and genes and gene related sequences 40% of genome
Characteristics of the human genome
Many introns in human genes
A gene can I code several proteins with posttranscriptional mechanisms accounting for the observed protein diversity in humans
Types of DNA sequence variation
Two types of common genetic variation (polymorphisms in DNA sequence): single nucleotide polymorphisms (SNPs): inherited variations involving a single base originating by point mutations
Short tandem repeats(STRs) short repetitive sequences occurring side by side on chromosomes, usually in noncoding regions
Genetic markers
Polymorphisms are used as genetic markers in analyses of humans and other organisms
-sequence differences (alleles) in genetic markers must be identifiable by current DNA analysis methods; codominance is best
-restriction enzymes can be used to identify SNPs (an insertions and deletions) in restriction sites. SNPs can be in toy assayed
-STRs vary in length and there are multiple alleles at each marker locus
Gel Electrophoresis
DNA fragments can be separated by size using gel electrophoresis.
A mixture of fragments is placed in a well of semisolid gel. An electric field is applied across the gel
Negatively charged DNA fragments move towards positive end.
Smaller fragments move through the gel faster than larger ones
Restriction Fragment Length Polymorphisms (RFLPs)
Restriction enzymes cut DNA at specific sequences generating smaller fragments. Mutations at restriction sites that change the ability of the enzyme to cut can be assayed as RFLPs. These are observed as bands on electrophoresis gel following digestion of PCR-amplified target or hybridization of digested DNA with probe.
Genotype to phenotype
Mendelian(or discrete) traits:
-single gene, affecting discrete phenotypic differences
Phenotypic variation is often complex:
-phenotypes vary continuously over a range- quantitative variation
-phenotypic variation is usually due to the action of multiple genes and also influenced by the environment
Darwinian selection LO
Darwin outlined basic principles and supporting evidence that provide a foundation for modern evolutionary theory
Populations evolve through differential survival And reproduction of individuals
Natural selection influences a population through individual differences and fitness Xpress through survival and reproduction connection.
Variation and populations LO
Random combination of alleles to form genotypes of asexual population is modeled by the hardy Weinberg equations enter nonrandom mating can cause a deviation between observed genotype frequencies from the expectations of the hardy