Genetics Final

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DNA created by joining together pieces of DNA from different sources

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Recombinant DNA

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Message use to copy or clone DNA and often known as gene splicing in the earlier days, marked a major advance and research and molecular biology and genetics, allowing scientists to isolate and study specific DNA sequences.

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Recombinant DNA technology

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DNA cutting enzymes

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Restriction enzymes

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Two important tools used to construct an amplify recombinant DNA molecules

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Restriction enzymes and DNA cloning vectors

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A restriction enzyme recognizes and binds to DNA at a specific nucleotide sequence

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Recognition sequence or restriction site

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a nucleotide sequence that reads the same on both strands of the DNA when read and the five prime to 3 prime direction. Mom, dad, pop, bob, ect.

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Palindrome

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Enzyme such as Eco RI and HindIII make offset cuts in the DNA strands, thus producing fragments with single-stranded overhanging ends

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Cohesive ends or sticky ends

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Alu I and Bal I cut both strands at the same nucleotide pair, producing DNA fragments with double-stranded ends

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Blunt ends

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When mixed together, single-stranded ends of DNA fragments from different sources cut with the same restriction enzyme can kneel, or stick together, but hydrogen bonding of complementary base pairs and single-stranded ends.

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Sticky ends sticking together

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DNA fragments will seal the phosphodiester backbone of the DNA to Covalently join the fragments together to form recombinant DNA molecules.

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DNA ligase

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Do you need molecules that accept DNA fragments and replicate these fragments when vectors are introduced into host cells

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Cloning vectors

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To distinguish host cells that have taken up the actors from host cells that have not, the vectors should carry a

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Selectable marker gene (usually an antibiotic resistant gene)

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Genetically modified bacterial ______________ were the first vectors developed, and they are still widely used for cloning. they were derived from naturally occurring plasmids.

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plasmids

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Postmans are introduced into bacterial by the process of ______________. Two main techniques

The first technique involves treating cells with calcium ions and using a brief heat shock to pulse DNA into cells. The other techniques, is called electroporation, uses a brief, but high intensity, pulse of electricity to moved in a bacterium to bacterial cells.

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Transformation

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Plasmid vector have also been genetically engineered to contain a number of restriction sites for commonly used restriction enzymes in a region called the _______ _________ ________. They allow scientists to clone a range of different fragments generated by many commonly-used restriction enzymes.

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Multiple cloning site

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Genes that provide resistance to antibiotics such as ampicillin and jeans such as lacZ Jean are very effective selectable marker genes. This process is referred to as_____-__________ _____________ for a reason that will soon become obvious. in this process a plastid is used that contains the lacZ gene incorporated into the multiple cloning site the lacZ gene encodes the enzyme B-galactosidase, which, as you aren't earlier in the text, is used to clean the disaccharide lactose Intuit component monosaccharides glucose and galactose. this process takes advantage of the enzymatic activity of B-galactosidase.

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Blue - white selection

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Two other examples of vectors that can be used to clone large fragments and DNA are

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Bacterial artificial chromosomes and yeast artificial chromosomes

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Rhizobium contains a plasmid called the ___ _______________.

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Time plasmid (tumor-inducing)

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1.) yeast is eukaryotic organisms, it can be grown and manipulated in much the same way as bacterial cells.

2.) the genetics of yeast has been in tensively studied, providing a large catalog of mutants and a highly developed genetic map.

3.) entire yeast genome has been sequence, and scenes in organism organism have been identified.

4.) to study the function of eukaryotic proteins it is necessary to use a hose so that can modify the protein, by adding carbohydrates, for example after it has been synthesized, to convert it to a functional form.

5.) used has been used for centuries in the baking and brewing industries and is considered to be a safe organism for producing proteins for vaccines and therapeutic agents.

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Reasons for choosing yeast as cloning vectors

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Represent a collection of cloned DNA

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DNA libraries

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Consists of many overlapping fragments of the genome, with at least one copy of every DNA sequence in an organism's genome, which in summary span the entire genome. In making this library, DNA is extracted from cells or tissues and cut randomly with restriction enzymes, and the resulting fragments are inserted into vectors using techniques that we discussed in the previous section.

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Genomic library

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Offer certain advantages over genomic libraries and continue to be useful methodology for gene cloning. This library contains DNA copies called cDNA, which are made from mRNA molecules isolated from cultured cells or a tissue sample and therefore represent the genes being expressed in cells at the time the library was made.

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Complementary DNA library

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This enzyme Extends the oligo (dT) primer and synthesizes a complementary DNA copy of the mRNA sequence. the product of this reaction is an mRNA / DNA double-stranded hybrid molecule

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Reverse transcriptase

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Several methods allow us to sort through a library and isolate specific genes of interest, in this approach is called

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Library screening

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Is any DNA or RNA sequence that is complementary to some part of a cloned sequence present in the library - the target gene or sequence to be identified.

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Probe

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Is the nucleotide sequence of any of the DNA on the membrane is complementary to the probes, the double-stranded DNA - DNA hybrid molecule will form, one strand from the probe and the other from the cloned DNA on the membrane. This is called

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Hybridization

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Is a rapid method of DNA cloning that extends the power of recombinant DNA research and many cases eliminates the need to use host cells for cloning.

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Polymerase chain reaction

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1. Colonies of the library are overlaid with a dna-binding membrane such as nylon.

2. Colonies are transferred to membrane, then lysed, and DNA is denatured

3. Membrane is placed in a heat sealed bag with a solution containing the labeled probe - the probe hybridizes with denatured DNA from colonies

4. Membrane is rents to remove excess sperm, then dried - x-ray film is placed over the filter for autoradiography or chemiluminescence signal from the probe recorded with a digital camera.

5. Using the original plate, cells are pic from the colonies that hybridize to the probe

6. Cells are transferred to a medium for growth and further analysis

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Steps of PCR

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Number of DNA molecules in a reaction doubles during the

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Cycle

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the double-stranded DNA to be cloned is denatured into single strands. The DNA can come from many sources, including genomic DNA, mummified remains, fossils, or forensic samples such as dried blood or semen, single hairs, or dried samples from medical records. heating 292 - 95 degrees Celsius for about 1 minute denatures the double-stranded DNA into single strands.

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Denaturation

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The temperature of the reaction is lowered to a temperature between 45 degrees and 65 degrees Celsius, which causes the primer binding to the denatured, single-stranded DNA. as described earlier, the primers are short oligonucleotides complementary to sequences flanking the target DNA. the primers serve as a starting point for DNA polymerase to synthesize new DNA strands complementary to the target DNA. factors such as primer lane, base composition of primers, and whether or not all bases and a primer complementary to bases and the target sequence are among primary consideration when selecting a hybridization temperature for an experiment.

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Hybridization / annealing

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The reaction temperature is adjusted between 65 degrees and 75 degrees Celsius, and DNA polymerase uses the primer as a starting point to synthesize new DNA strands that adding nucleotides to the ends of the primers in a five prime to 3 prime direction.

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extension

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RNA is isolated from cells or tissues to be studied, and reverse transcriptase is used to generate double-stranded cDNA molecules. this reaction is followed by PCR to amplify cDNA with a set of primers specific for the gene interest. amplified see DNA fragments are then separated and visualized on in agarose gel. This process can be used to evaluate relative levels of gene expression in different samples.

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Reverse transcriptase PCR

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This approach makes it possible to determine the amount of PCR product made during an experiment which enables researchers to quantify implication reactions as they occur in real time.

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Quantitative real-time PCR

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Establishes the number of, order of, and distance between restriction enzyme cleavage sites along a cloned segment of DNA, thus providing information about the length of the cloned and cert and the location of restriction enzyme cleavage sites within the clone.

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Restriction maps

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Used to identify which lens in a library contain a given DNA sequence and to characterize the size of the fragments

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Southern blot

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RNA blotting is referred to as

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Northern blotting

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Is widely used techniques for analyzing proteins

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Western blotting

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Is a powerful tool that involves hybridizing aprove directly to a chromosome or RNA without blotting. Can be carried out with isolated chromosomes on a slide or directly In situ and tissue sections or entire organisms, particularly when embryos are used for various studies and developmental genetics

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Fluorescent in situ hybridization fish

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individual chromosomes can be detected using probes labeled with the dyes that will fluoresce at different wavelengths.

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Spectral karyotypes

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In this technique, a double-stranded DNA molecule whose sequence is to be determined is converted into single strands that are used as a template for synthesizing a series of complementary strands.

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Dye deoxy nucleotide change termination sequencing or Sanger sequencing

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To disrupt or eliminate a specific gene or genes of interest.

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Knockout gene

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One popular approach involves using electroporation to deliver the vector into _______ _____ ______ grown in culture.

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Embryonic stem cells

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Allow one to control in a target gene is disrupted. Used If no mice for a particular gene of interest cannot be dry by traditional knock out approaches.

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Conditional knockout

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Express or often overexpress a particular gene of interest, the transgene.

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Transgenic animals, also called knockin animals

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The complete set of DNA in a single cell of an organism.

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Genome

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The study of genomes , by using a newly developed method of DNA sequencing to sequence the 5400 gosh nucleotide genome of a virus.

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Genomics

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Focuses on sequencing genomes and analyzing nucleotide sequences to identify genes and other important sequences such as gene regulatory regions

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Structural genomics

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Most widely used strategy for sequencing and assembling an entire genome. Genomic DNA is cut into fragments and contigs are made , entire chromosome is assembled by computer program, and pregnant airline based on identical DNA sequences.

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Whole-genome sequencing, also known as shotgun cloning

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Many of the early computer automated sequencers, designed for this sequencing, could process millions of base pairs in a day. B sequencers contained multiple capillary gels that are several feet long.

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High-throughput sequencing

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The use of computer hardware and software on my monday applications to organize, share, analyze data related to gene structure, gene sequence and expression, and protein structure and function.

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Bioinformatics

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One of the largest genomic databases. It shares and acquires data from databases in Japan and Europe, it contains more than a hundred 50 billion bases of sequence data from over a hundred thousand species.

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GenBank

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Relies heavily on bioinformatics, & a wealth of different software tools are available to carry it out. when an issue approach is to compare the newly sequence genomic DNA to the known sequences already stored in various databases.

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annotation

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The NCBI provides access to _________, a very popular software application for searching through banks of DNA and protein sequence data.

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Blast , basic local alignment search tool

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BLAST searches calculate an identity value, which is determined by the sum of identical matches between aligned sequences divided by the total number of bases aligned

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Similarity score

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The blast report also provides an expected value, based on the number of matching sequences in the database that would be expected by chance.

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E - value

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is the study of gene functions, based on the resulting Arnaiz or possible protein stay in code, and the functions of the other components of the genome, such as gene regulatory elements.

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Functional genomics

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Genes that are evolutionarily related

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homologous genes

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Is homologous genes in different species are thought to have descended from a gene in a common ancestor the gene is known as

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Orthologs

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Homologous genes in the same species are called

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Paralogs

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Identification of ____________ __________, such as ion channels, membrane spanning regions, dna-binding regions, secretion and export signals , and other structural aspects of a polypeptide that are encoded by a DNA sequence, can in turn be used to predict protein function.

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Protein domains

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The structure of many DNA proteins have characteristic patterns or ______________, such as the helix turn helix, leucine zipper, or zinc finger motif

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motifs

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Most coordinated international efforts to determine the sequence of the human genome and to identify all the genes that it contains.

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Human Genome Project

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The number of genes is much lower than the number of predicted proteins in part because many genes code for multiple proteins through

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Alternative splicing

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All humans of all racial and ethnic origins are genetically 99.9% similar, humans only differ by

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Single nucleotide polymorphisms and copy number variations

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The analysis of all the proteins in a cell or tissue

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Proteomics

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The analysis of proteins and enzymatic pathways involved in cell metabolism

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Metabolomics

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The analysis of the carbohydrates of a cell or tissue

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Glycomics

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The analysis of the effects of toxic chemicals on genes, including mutations created by toxins and changes in gene expression caused by toxins

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Toxicogenomics

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The analysis of genomes of organisms collected from the environment

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Metagenomics

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The development of customized medicine based on a person's genetic profile for a particular condition

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Pharmacogenomics

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The anylysis of all expressed genes in a cell or tissue

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transcriptomics

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Focuses on understanding the interaction between diet and genes

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Nutritigenomics

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Generating fascinating data from meniscal amounts of ancient DNA obtained from bone in other tissues such as hair that are tens of thousands to about 700,000 years old, and often involve samples from extinct species.

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Stone Age genomics

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George church Harvard and his calling recruited volunteers to provide DNA for individual genome sequencing on the understanding that the genome data will be made publicly available

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Personal Genome Project

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A main goal of encode was to use both experimental approaches including chip - based methods and bioinformatics to identify and analyze functional elements of the genome, such as transcriptional start sites, promoters, and enhancers, which regulate the expression of human genes

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Encyclopedia of DNA elements project

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To complete the genome of an estimated 600 to 1000 microorganisms, bacteria, viruses, and yeast live on an inside humans

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human Microbiome project

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Compares the genomes of different organisms to answer questions about genetics and other aspects of biology

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Comparative genomics

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gene density. in prokaryotes, Jean density is close to 1 jam / kilobase. And eukaryotic genomes, there is a wide range of gene density. and yeast, there is about one gene / 2 Kb, in Drosophila about one gene / 13 Kb, and then humans, gene density varies greatly from chromosome to chromosome. human genome 22 has about 1 gene / 64 Kb well Chrome is in 13 has one gene / 155 KB of DNA

Introns. most eukaryotic genes contain introns

Repetitive sequences. The presence of introns and the existence of repetitive sequences are two major reasons for the wide range of genome size in eukaryotes

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Eukaryotic genomes have several features not found in prokaryotes

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the human and Neanderthals genomes are _____ similar

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99%

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Groups in genes that share similar but not identical DNA sequences through duplication and descent from a single ancestral gene.

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Multigene family

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A group of related multigene families

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Superfamily

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Is members in code very similar but not identical polly peptide chains with closely related functions

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Globin gene superfamily

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is widely used because it enables researchers to analyze all of a samples expressed genes simultaneously

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DNA microarray analysis

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DNA microarray analysis also known as, ______ ______, consist of a glass microscope slide onto which single stranded DNA molecules are attached, or spotted, using a computer controlled high speed robotic arm called arrayer.

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Gene chips

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The complete identification, characterization, and quantitative analysis of the proteome of a cell, tissue, or organism - can be used to reconcile differences between the number of genes in a genome in the number of different proteins produced.

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Proteomics

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Defined as evolutionary change within populations of a species.

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Microevolution

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Defined as evolutionary events leading to the emergence of new species and other taxonomic groups

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Macroevolution

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Is a group of individuals belonging to the same species that live in a defined geographic area and actually or potentially in a interbreed

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Population

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The genetic information carried by members of a population

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Populations gene pool

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One way to determine whether genetic variation exist in a population is to use artificial selection. If genetic variation is present, the phenotype will change every few generations. The most direct way to estimate genetic variation is to compare the nucleotide sequence of genes carried by individuals in a population.

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Genetic variation

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Proposed by motoo kimura in 1968, proposes that mutations leading to amino acid substitution are usually detrimental, within only a very small fraction being favorable. Some mutations are neutral, that is they are functionally equivalent to be a Leo they replace. mutations that are favorable or detrimental are preserved or removed from the population, respectively, by natural selection. however, the frequency of neutral alleles in a population will be determined by mutation rates and random genetic drift, and not by selection.

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The neutral theory of molecular evolution

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Describes what happens to alleles and genotype frequencies in an ideal population that is infinitely large and randomly mating, and that is not subject to any evolutionary forces such as mutation, migration, or selection. To predictions of the model is the frequency of alleles in the gene pool do not change over time and if two alleles at a locus, A and a , are considered, then as we will show later in this chapter, after one generation of random mating, the frequency of genotypes AA:Aa:aa in a population can be calculated as p2 2pq+q2

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Hardy Weinberg law

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2 methods of hardy-weinberg the wall

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Individuals of a species exhabit variations and phenotype, many of these variations, even small and seemingly insignificant ones, are heritable and passed on to offspring, organisms tend to reproduce in an exponential fashion. More offspring are produced than can survive. This causes members of a species to engage in a struggle for survival, competing with other members of the community for scarce resources, and in the struggle for survival, individuals with particular phenotypes will be more successful than others, allowing the former to survive and reproduce at higher rates.

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Natural selection

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an individual organism's genetic contribution to future generations

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Fitness

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At one end of the spectrum present in the population become selected for or against usually as a result of changes and the environment.

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Directional selection

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Tends to favor intermediate phenotypes, with those at both extremes being selected against

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Stabilizing selection

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Selection against intermediate and for both phenotypic extremes

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Disruptive selection

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Alone acts to create new alleles

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Mutation

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Occurs when individuals move between populations

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Migration

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In small populations, significant random fluctuation in allele frequencies are possible by chance alone, situation known as ..... The degree of fluctuation increases as the population size decreases

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Genetic drift

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To small population size, drift can rise through this effect, which occurs when a population originates from a small number of individuals. Although the population me later increased to a larger size, the genes carried by all members are derived from those of the founders

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Founder effect

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Develop on a large population undergoes a drastic but temporary reduction in numbers. even though the population recovers, its genetic diversity has been greatly reduced

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genetic bottleneck

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Simular phenotypes are more likely to make dissimilar ones

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positive assortive mating

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Occurs when dissimilar phenotypes are more likely to mate

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negative assortive mating

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The form of nonrandom mating most commonly found to affect genotype frequencies in population genetics . occurs when mating individuals are more closely related than any two individuals drawn from the population at random.

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Inbreeding

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Can be defined as a group of actual or potentially interbreeding organisms that is reproductively isolated and nature from all other such groups

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Species

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biological barriers that prevent or reduce interpreting between populations . these mechanisms may be ecological, behavioral, seasonal, mechanical, or physiological

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Reproducive isolating mechanisms

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Prevent individuals from mating in the first place. Individuals from different populations may not find each other at the right time, may not recognize each other as suitable mates, or maybe try to find me but find that they are unable to do so.

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Prezygotic isolating mechanisms

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Create reproductive isolation even when the members of two populations are willing and able to mate with each other for example, genetic divergence may have reached the stage where the viability or fertility of hybrids is reduced. Hybrid zygotes may be form, but all or most may be enviable.

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Postzygotic isolating mechanisms

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technology that allows the recovery and genomic sequencing of DNA from fossil humans.

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Paleogenomics

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Number of plant and animal species in the ecosystem

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Interspecific

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Intra or inter population genetic diversity, measures poly morphic loci

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Intraspecific

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Reduction in population size, loss of habitat, population fragmentation, and agricultural processes

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Loss of genetic diversity

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Alloy me analysis, DNA profiling (VNTRS and STRs) can resolve down to one base pair difference, mitochondrial and chloroplast DNA analysis, RFLP studies, pcr-based DNA fingerprinting

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Identifying genetic diversity

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Is the exchange of alleles between two or more populations

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Gene flow

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Reduction in genetic diversity, loss of important alleles, and reduction in heterozygousity

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genetic erosion

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Ex situ conservation, gene banks, in situ conservation, population augmentation( genetic swamping, out breeding depression and loss of Co-adapted gene complexes.

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Conservation of genetic diversity