Nucleic Acid Technology

 

Cloning Genes–Overview

Overview of the cloning process used to isolate and amplify individual genes.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 15 (4:21 - 10:19)

Brief introduction to the need to clone genes, the use of restriction enzymes to cut DNA, and PCR to amplify DNA.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 24 (3:20 - 9:40)

Upward arrow. Back to Top

Cloning–Restriction Enzymes

Discovery and function of restriction enzymes used in cloning. Restriction enzymes cleave DNA at certain palindromic restriction sites/sequences.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 15 (10:19 - 31:20)

Upward arrow. Back to Top

Cloning–DNA Ligases

Ligases are used to rejoin pieces of DNA cut by restriction enzymes. DNA pieces with overhangs must have complementary ends to ligate together.

Course Material Related to This Topic:

Upward arrow. Back to Top

Cloning–Vectors/Plasmids

Vectors are circular pieces of DNA that can carry genes into the bacteria. Vectors often contain origin of replication, restriction sites, drug resistance, and bacterial promoters.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 15 (34:04 - 44:04)

Upward arrow. Back to Top

Cloning–Transformation and Selection

Bacteria randomly take up plasmids in the environment. Select for transformed bacteria that have taken up plasmids using antibiotic resistance such as ampicillin and kanamycin.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 15 (44:04 - 50:10)

Upward arrow. Back to Top

Cloning by Complementation

Find clone of interest in the library by complementation. Transform plasmids into mutant bacteria deficient in the gene and look functional rescue.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 16 (10:32 - 19:56)

Cloning a single gene by rescuing the function of the mutant using a library.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 24 (36:51 - 40:56)

Upward arrow. Back to Top

Cloning by Sequence

DNA sequence can be inferred from the purified and sequenced protein. Radioactive complementary DNA probes are used to hybridize to the clone of interest.

Course Material Related to This Topic:

Upward arrow. Back to Top

Cloning by Position I and II

Map the gene of interest based on pedigrees. Use recombination rates to determine linkage between gene of interest and other genetic markers (small variations in the genome) within each family.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 16 (41:40 - 50:32)
  • Watch {}video clip from Lecture 17 (2:13 - 8:36)

Upward arrow. Back to Top

Expression Library

A collection of human mRNA can be reverse transcribed into a cDNA library and used in cloning. Bacteria transformed with cDNA can make human proteins.

Course Material Related to This Topic:

Upward arrow. Back to Top

Restriction Mapping and Gel Electrophoresis

Plasmids are purified and clones are redigested with multiple restriction enzymes and the size is determined by gel electrophoresis. Analyze clones by determining the location of the restriction sites.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 17 (21:44 - 30:02)

Upward arrow. Back to Top

Sequencing Genome

Genomes can be sequenced by sequencing smaller, approximately 1000bp long, segments and combining them by overlapping regions.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 18 (1:39 - 7:32)

Upward arrow. Back to Top

Polymerase Chain Reaction (PCR)

PCR is a form of in vitro DNA replication used to amplify DNA segments. Primers are designed to amplify small segments of DNA. Thermostable Taq polymerase is used so it does not denature at high temperatures.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 18 (19:09 - 33:15)

Uses of PCR in forensics and familial diseases. Components needed for PCR-template, nucleotides, primers, and heat resistant polymerase. Steps of PCR done at various temperatures-denature double helix, anneal primers, elongate chain.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 25 (21:09 - 31:50)

Upward arrow. Back to Top

PCR–Application

PCR has low fidelity. The process can be used to look for integrated virus segments or cancer cell. Other applications include: Single sperm typing, embryotyping, and forensics.

Course Material Related to This Topic:

Upward arrow. Back to Top

Cloning Genes–Steps

General outline of the steps, components, and design of cloning.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 24 (9:40 - 15:59)

Upward arrow. Back to Top

Restriction Enzymes I and II

Course Material Related to This Topic:

Discussion of restriction enzymes, palindromic restriction sites, "sticky" ends, and ligation. History of Luria's experiment that discovered of restriction enzymes in bacteria. Example: EcoRI.

  • Watch {}video clip from Lecture 24 (15:59 - 22:24)

Clarification of restriction enzymes, cleavage, sticky ends, and 3' OH and 5' phosphate. Linkage of the complementary sticky ends.

  • Watch {}video clip from Lecture 26 (0:00 - 4:32)

Upward arrow. Back to Top

Discovery of Restriction Enzymes

Bacteriophage experiment that discovered restriction enzymes. Bacteria that have restriction enzymes protect their own DNA by methylating their restriction sites.

  • Watch {}video clip from Lecture 24 (26:49 - 33:44)

Upward arrow. Back to Top

Recombinant DNA–Libraries I and II

Course Material Related to This Topic:

Steps in constructing a DNA library. Cut genomic DNA into segments, insert into vectors, and transform vectors into bacteria.

  • Watch {}video clip from Lecture 24 (33:44 - 36:51)

Constructing DNA library by cutting up a genome and inserting pieces into vectors. The vector containing the gene of interest can be selected by rescuing functional defects (complementation).

  • Watch {}video clip from Lecture 26 (18:15 - 21:59)

Upward arrow. Back to Top

Gel Electrophoresis and Restriction Maps

Principles and uses of gel electrophoresis. Analysis of clones using restriction enzymes and gel electrophoresis. Image of gel apparatus and UV image.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 25 (1:53 - 10:25)

Upward arrow. Back to Top

DNA Sequencing I and II

Course Material Related to This Topic:

Sequencing accomplished by using dideoxynucleotide units that stop DNA polymerization in the middle of the chain. Colored dyes and gel electrophoresis are used to determine the length of the fragments and sequence of bases.

Review and clarification of DNA sequencing. Brief but good explanation.

  • Watch {}video clip from Lecture 27 (0:00 - 3:04)

Upward arrow. Back to Top

Vectors/Plasmids

Circular vectors with origin of replication are used as vehicles to carry linear DNA into bacteria.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 26 (5:12 - 9:05)

Upward arrow. Back to Top

Recombinant DNA for Eukaryotes I and II

Course Material Related to This Topic:

Issues with cloning eukaryotic genes using bacterial plasmids-different promoters and introns.

  • Watch {}video clip from Lecture 26 (21:59 - 25:16)

cDNA libraries are created from mRNA to DNA, so the library does not contain introns. Fuse mammalian gene with E. coli promoter, so E. coli can make human proteins. Fuse mammalian promoters with reporter protein (can be easily assayed) such as Beta-galactosidase and GFP.

  • Watch {}video clip from Lecture 27 (14:00 - 23:40)

Upward arrow. Back to Top

Cloning by Hybridization/Sequence

Synthesize the DNA using the protein sequence and radioactive P32. Hybridize DNA probe to recombinant library.

Course Material Related to This Topic:

  • Watch {}video clip from Lecture 26 (25:16 - 34:54)

Upward arrow. Back to Top

Cloning by Position

Cloning by linkage to genetic markers such as single nucleotide polymorphisms and recombination.

Course Material Related to This Topic:

Upward arrow. Back to Top

Restriction Enzymes

Introduction to restriction enzymes as a nucleic acid technology.

Course Material Related to This Topic:

Upward arrow. Back to Top

Gel Electrophoreisis

Introduction to gel electrophoresis as a nucleic acid technology.

Course Material Related to This Topic:

Upward arrow. Back to Top

DNA Transformation

Course Material Related to This Topic:

Introduction to bacterial transformation and plasmids.

A continuation of the discussion on the method of bacterial transformation, use of plasmids, and cloning, including clone identification and cloning by complementation.

Course Material Related to This Topic:

Upward arrow. Back to Top

Polymerase Chain Reaction

Discussion of the recent development of PCR.

Course Material Related to This Topic:

Upward arrow. Back to Top

Discovery of Restriction Enzymes–Luria Experiment

Luria's experimental design and results that led to the discovery of restriction enzymes in bacteria.

Course Material Related to This Topic:

Upward arrow. Back to Top

Yeast and Genetic Studies

Yeast as a model organism. Techniques and experiments used in the making, identifying, and characterizing yeast mutants. Test of Recessivity, Complementation Test and Epistasis Test.

Course Material Related to This Topic:

Upward arrow. Back to Top

Temperature Sensitive Mutants

Brief explanation of the use of temperature sensitive mutants to study essential genes.

Course Material Related to This Topic:

Upward arrow. Back to Top

Restriction Maps and Expression Vector

Creating restriction maps using restriction enzymes. Necessary components of a bacterial expression vector that carry a gene of interest.

Course Material Related to This Topic:

Upward arrow. Back to Top

Recombinant DNA–Example

Steps involved in expressing a C. elegans gene in bacteria.

Course Material Related to This Topic:

Upward arrow. Back to Top

Recombinant DNA and Cloning

Steps involved in expressing a C. elegans gene in bacteria.

Course Material Related to This Topic:

Upward arrow. Back to Top

Protein Gel

Purification and separation by gel electrophoresis of proteins produced in bacteria. Native versus denaturing gels.

Course Material Related to This Topic:

Upward arrow. Back to Top

Mutant Hunt

Steps and requirements to clone a functional gene by growing mutant yeasts in various mediums.

Course Material Related to This Topic:

Upward arrow. Back to Top

Complementation–Conceptual Questions

Basics and uses of a complementation test to characterize a group of mutants with the same phenotype.

Course Material Related to This Topic:

Upward arrow. Back to Top

Complementation–Examples (Phe and Lysis Pathways)

Examples of complementation groups for mutants and use of complementation to establish a biosynthetic pathway.

Course Material Related to This Topic:

Upward arrow. Back to Top

Epistasis–Examples

Examples of complementation groups for mutants and use of complementation to establish a biosynthetic pathway.

Course Material Related to This Topic:

Upward arrow. Back to Top

Cloning–Tools

Definition and explanation of cloning, vector, library, restriction enzyme, and recombinant DNA technology.

Course Material Related to This Topic:

Upward arrow. Back to Top

Cloning–Application

Example of inserting a gene into a vector using restriction enzymes and ligase. Transform vector into bacteria and select using drug resistance. Analyze clone using restriction enzymes and gel electrophoresis.

Course Material Related to This Topic:

Upward arrow. Back to Top

Cloning–Library Construction and Cloning by Complementation

Steps in making a genomic library of wild type yeast that can be used in cloning by complementation to rescue mutant yeast.

Course Material Related to This Topic:

Upward arrow. Back to Top

Recombinant DNA Technology and Forensics

Using DNA to solve crimes. Recombinant DNA technology used include: DNA sequencing, restriction digest, and gel electrophoresis.

Course Material Related to This Topic:

Upward arrow. Back to Top

Molecular Cloning

Constructing wild type E. coli library using restriction enzymes and plasmids. Cloning by complementation by rescue tryptophan biosynthesis deficiency.

Course Material Related to This Topic:

Upward arrow. Back to Top

Mutant Proteins

Analysis using gel electrophoresis. Mutations leading to changes in side chains that affect the function of the protein.

Course Material Related to This Topic:

Upward arrow. Back to Top

Complementation and Epistasis

Example of biochemical pathway with mutations in several complementation groups.

Course Material Related to This Topic:

Upward arrow. Back to Top

Genomic Library

Reagent and enzymes needed to construct a genomic library.

Course Material Related to This Topic:

Upward arrow. Back to Top

Restriction Mapping

Mapping restriction sites in a gene or vector using restriction enzymes and gel electrophoresis.

Course Material Related to This Topic:

Upward arrow. Back to Top

Expression in Bacteria

Modifications used to express eukaryotic genes in bacteria.

Course Material Related to This Topic:

Upward arrow. Back to Top