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Applied molecular genetics

By Judith Hart,2014-08-30 00:13
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Applied molecular genetics

    目录

    Lecture 1 - Overview of AMG Download PDF file .............................................4 Lecture 2 - Nucleic Acid Biochemistry ............................................................. 13 Lecture 3 - Nucleic Acids Lab Practicums......................................................... 27 Lecture 4 - E. coli Hosts and Plasmid Biology .................................................. 39 Lecture 5 - Bacteriophage and Lab Practicum 2 ................................................ 61 Lecture 6 - DNA Libraries and Mutagenesis ..................................................... 76 Lecture 7 - Genomics and Genome Mapping .................................................... 94 Lecture 8 - Genome Mapping Lab Practiums .................................................. 115 Lecture 9 - Biochemistry of cDNA Synthesis .................................................. 129 Lecture 10 - Screening cDNA libraries............................................................ 147 Lecture 11 - cDNA Cloning Lab Practicums ................................................... 161 Lecture 12 - The Polymerase Chain Reaction (PCR) ....................................... 172 Lecture 13 - Reverse Transcriptase-Mediated PCR ......................................... 188 Lecture 14 - Use of PCR in Diagnostics and Research .................................... 198 Lecture 15 - Gene Expression in Cultured Cells .............................................. 220 Lecture 16 - Yeast Models and Protein Expression .......................................... 245 Lecture 17 - Gene Expression Lab Practicum .................................................. 258 Lecture 18 - Transgenic Flies and Crop Plants ................................................ 277 Lecture 19 - Mouse Transgenesis .................................................................... 296 Lecture 20 - Transgenic Livestock and Animal Cloning .................................. 320 Lecture 21 - Highthrougput Instrumentation ................................................... 330 Lecture 22 - Computer Lab and Website Basics .............................................. 345 Lecture 23 - Bioinformatics and Proteomics ................................................... 348 Lecture 24 - DNA Forensics and Pharmacogenomics ...................................... 371 Lecture 25 - Gene Therapy and Stem Cell Technology .................................... 380

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    Section 1 - Laboratory Techniques

    Biochemistry of Applied Molecular Genetics

    o Overview of Applied Molecular Genetics .......................................Lecture 1

    o Nucleic Acid Biochemistry .............................................................Lecture 2

    o Homework 1 Questions

    o HW-1 Answer Key (pdf file)

    o Nucleic Acids Lab Practicums .........................................................Lecture 3 Laboratory Tools for Molecular Genetics

    o E. coli K-12 ; Plasmid biology ........................................................Lecture 4

    o Homework 2 Questions

    o HW-2 Answer Key (pdf file)

    o Bacteriophage vectors; pET expression systems ..........................Lecture 5

    o DNA libraries; In vitro mutagenesis ...............................................Lecture 6

    o Homework 3 Questions

    o HW-3 Answer Key (pdf file)

    Section 2 - Core Methods Genome Structure and Function

    o Overview of Genomics; Gene Mapping ........................................Lecture 7

    o Gene Mapping Lab Practicums .....................................................Lecture 8

    o Homework 4 Questions

    o HW-4 Answer Key (pdf file)

    Isolation and Characterization of Gene Transcripts

     Biochemistry of cDNA Synthesis. ........................................Lecture 9

     Screening cDNA Libraries ...................................................Lecture 10

     Homework 5 Questions

     HW-5 Answer Key (pdf file)

     cDNA cloning Lab Practicums ............................................Lecture 11

    Mid-term Exam Answer Keys 471a PDF and 571a PDF

    The Polymerase Chain Reaction

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    o Biochemistry of the PCR Reaction .............................................Lecture 12 o Reverse Transcriptase-Mediated PCR .......................................Lecture 13 o PCR Applications and Lab Practicum ..........................................Lecture 14

    Section 3 - Specialized Applications

    Expression of Cloned Genes in Cultured Cells

    o Gene Expression in Cultured Cells ............................................Lecture 15 o HW-6 Answer Key (pdf file)

    o Yeast as a Model; Protein Expression in Cultured Cells ...............Lecture 16 o Gene Expression Lab Practicum ..................................................Lecture 17 o Homework 7 Questions

    o HW-7 Answer Key (pdf file)

    Construction of Transgenic Organisms

    o Transgenic Flies and Plants ........................................................Lecture 18 o Mouse Transgenesis .................................................................Lecture 19 o Homework 8 Questions

    o HW-8 Answer Key (pdf file)

    o Nuclear Transfer and Animal Cloning............................................Lecture 20 Functional Genomics and Bioinformatics

    o Highthroughput instrumentation ..................................................Lecture 21 o Computer Lab and Website Basics ...........................................Lecture 22 o Bioinformatics and Proteomics ..................................................Lecture 23 o Homework 9 Questions

    o HW-9 Answer Key (pdf file)

    Medical Molecular Genetics

    o DNA Forensics and Pharmacogenomics ..................................Lecture 24 o Gene Therapy and Stem Cell Biology .......................................Lecture 25 o Homework 10 Questions

    o HW-10 Answer Key (pdf file)

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    Lecture 1 - Overview of AMG

    Download PDF file

    Flow of Genetic Information

    Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymers consist of repeating units of deoxynucleotides and ribonucleotides, respectively. With the exception of some viruses, most all organisms on this planet store their cellular blueprints for life in double-stranded DNA molecules called chromosomes. In eukaryotic cells,

    chromosomes are copied during cell division, recombined and shuffled as a result of sexual reproduction, and transcribed into complementary RNA molecules through a process called gene expression.

    Chemical information stored in the DNA coding sequences of a gene is transmitted to the protein synthesis machinery in the cell by mRNA “transcripts”. This relationship between

    the DNA, RNA and protein sequence information of a gene is sometimes referred to as the biochemical flow of genetic information.

    Describe three levels of genomic regulation with regard to cellular phenotype, for example, in response to extracellular stimuli, or as a result of terminal differentiation?

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Why is RNA instability a "good thing" in biological processes?

Chemical Composition of Nucleic Acids

    Each deoxyribonucleotide unit in DNA contains one of the four bases; guanine

    (G), adenine (A), cytosine (C) or thymine (T); RNA contains uracil (U) in

    place of thymine.

    Nature‟s ability to encrypt the essence of life in long strings of DNA sequence, is not a problem of storage space, but rather how accurately the cell machinery

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    is able to retrieve and interpret this vast amount of information. Applied molecular genetics is the exploitation of this knowledge to investigate and

    utilize the processes of DNA synthesis (replication), RNA synthesis

    (transcription) and protein synthesis (translation) to not only access, but also to manipulate, the information potential of organismal DNA.

DNA Replication

RNA and Protein Synthesis

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    What biochemical process in cells is characterized by formation of an RNA-DNA hybrid?

    Name an applied molecular genetic technique that depends on the formation of RNA-DNA hybrids.

    What accounts for the biochemical instability of RNA compared to DNA in laboratory research?

Structure of DNA

    The Biochemical basis of duplex stability is determined by base composition,

    ionic strength, length of duplex. Hydrogen bonds between purines and pyrimidines on opposing strands hold the double helix together. A-T and A-U

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    base pairs are contain 2 hydrogen bonds, whereas G-C base pairs have 3 hydrogen bonds.

    In addition to hydrogen bonding between complementary strands, the DNA helix is also stabilized by hydrophobic interactions between adjacent bases

    on the same strand (Van der Waals interactions). Although these "stacking"

    interactions do not contribute as much to helix stability as hydrogen bonding, it is important to note that "nearest neighbor" relationships contribute to complex nucleic acid structures such as those found in highly folded RNA.

    Transcription factors regulate initiation of RNA synthesis by binding to specific DNA sequences in gene promoters

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    Why do you think most transcription factors bind to bases in the major groove of DNA rather than to the minor groove?

    What "scientific" fact did Michael Crichton exploit to explain how dinosaurs could be "regenerated" using dinosaur DNA isolated from fossilized mosquitos in his fictional story Jurassic Park, i.e., can you tell what species a DNA fragment comes from by looking at the nucleotide composition?

Denaturation and Renaturation of Nucleic Acid Polymers

    The relative amount of single- or double-stranded DNA in solution can be experimentally determined using spectrophotometery to measure ultraviolet

    light absorbance at a wavelength of 260 nanometers (OD). The aromatic 260

    bases in DNA are less accessible to ultraviolet light in the double-stranded,

    compared to single-stranded form, which creates a measurable difference in

    the observed OD. Using this empirical difference in absorbance, it is 260

    possible to observe the effect of temperature on DNA structure by monitoring OD260 over a range of temperature range. The temperature at which 50% of the DNA is denatured is called the Tm or transition temperature.

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    The Transition Temperature (Tm) of a nucleic acid duplex is strongly effected by three factors;

1) Base composition

    2) Duplex length

    3) Ionic strength of the solution

    Two molecular genetic applications where extent of duplex formation is an

    important consideration are the use of short oligonucleotides in hybridization

    reactions, and heteroduplex formations between molecules that are less than

    100% complementary. The use of homologous, but not identical, DNA molecules in hybridization reactions is common when sequence divergence

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