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PAK 6-DNA Technology

Ch. 12-Molecular Genetics; Ch. 13-Genetics and Biotechnology sec. 2/3
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Clarissa Caro

on 11 April 2016

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Transcript of PAK 6-DNA Technology

The Human Genome
DNA Technology
Ch. 13-Genetics & Biotechnolgy
PAK 6 SEMINAR-DNA technology
Ch. 12-Molecular Genetics
Essential Questions:
Which experiments led to the discovery of DNA as the genetic material?
What is the basic structure of DNA?
What is the basic structure of eukaryotic chromosomes?
What is the role of enzymes in the replication of DNA?
How are leading and lagging strands synthesized differently?
How does DNA replication compare in eukaryotes and prokaryotes?
How are messenger RNA, ribosomal RNA, and transfer RNA involved in the transcription and translation of genes?
What is the role of RNA polymerase in the synthesis of messenger RNA?
How is the code of DNA translated into messenger RNA and utilized to synthesize a protein?
How are bacteria able to regulate their genes by two types of operons?
How do eukaryotes regulate the transcription of genes?
What are the various types of mutations?
DNA: The Genetic Material
The discovery that DNA is the genetic code involved many experiments.
Discovery of the Genetic Material
After the rediscovery of Mendel’s work, scientist began to look for the molecule involved in inheritance.
For many years, scientists struggled to determine if DNA or protein was the source of genetic information.
Discovery of Genetic Material
Griffith’s Experiment
First major experiment searching for the genetic material.
Involved transformation between two forms of S. pneumoniae
Set the stage for the search to identify the transforming substance
Avery’s Work
Identified the molecule that transformed the R strain of S. pneumoniae into the S strain
Concluded that when the S cells were killed, DNA was released
R bacteria incorporated this DNA into their cells and changed into S cells
Griffith's experiment
Discovery of the Genetic Material
Hershey and Chase’s Experiments
Used radioactive labeling to trace bacteriophage DNA and protein.
Concluded that the bacteriophage DNA was injected into the cell and provided the genetic information needed to produce new viruses.
Hershey and Chase Experiment
Nucleotides
Nucleotides are the subunits of nucleic acids, and consist of:
5-carbon sugar
Phosphate group
Nitrogenous base
DNA Structure
Chargaff
Analyzed the amount of A, G, T, and C in the DNA of various species
Chargaff’s rule: C = G and T = A
The structure question
Search for the structure of DNA was lead by four scientists:
Rosalind Franklin
, British chemist
Maurice Wilkins
, British physicist
The above scientists used X-ray diffraction techniques indicated that DNA was a double helix, or twisted ladder shape
Francis Crick
, British physicist
James Watson
, American biologist
DNA Structure
Watson and Crick
Using Franklin and Chargaff’s data, Watson and Crick measured the width of the helix and the spacing of the bases.
They built a model that conformed the Franklin and Chargaff’s data.
DNA structure
DNA often is compared to a twisted ladder.
Rails of the ladder are represented by the alternating deoxyribose and phosphate.
The pairs of bases (cytosine-guanine or thymine-adenine) form the steps.
Purine
base always binds to a
pyrimidine
base
DNA molecules have specific orientations of the two strands
On the top rail, the strand is said to be oriented 5’ to 3’.
The strand on the bottom runs in the opposite direction and is oriented 3’ to 5’.

Chromosome Structure
In prokaryotes, DNA molecules are contained in the cytoplasm, and consist mainly of a ring of DNA and associated proteins.
In eukaryotes, DNA is organized into individual chromosomes.
To fit into a cell, DNA coils around a group of beadlike proteins called histones.
DNA + histones form a nucleosome, which group together into chromatin fibers, which supercoil to form a chromosome.
DNA Structure & Function
http://www.teachertube.com/video/dna-structure-and-function-360133
Watch the link below:
Replication of DNA
DNA replicates by making a strand that is complementary to each original strand.
Semiconservative Replication
During semiconservative replication, parental strands of DNA separate, serve as templates, and produce DNA molecules that have one strand of parental DNA and one strand of new DNA.
Process occurs in three main steps: unwinding, pairing, and joining
Semiconservative Replication
Unwinding
DNA helicase, an enzyme, unwinds the helix, breaking the hydrogen bonds between bases
Single-stranded binding proteins keep the DNA strands separate during replication.
RNA primase adds a short segments of RNA primer, on each DNA strand.

Semiconservative Replication
Base pairing
The enzyme DNA polymerase adds appropriate nucleotides to the new DNA strand from the 3’ end.
The leading strand is built continuously, the lagging strand is built discontinuously in small segments called Okazaki fragments.
Semiconservative Replication
Joining
DNA polymerase removes the RNA primer and fills in the place with DNA nucleotides.
DNA ligase links the two sections.
Comparing DNA Replication in Eukaryotes and Prokaryotes
Eukaryotic DNA unwinds in multiple areas as DNA is replicated.
In prokaryotes, the circular DNA strand is opened at one origin of replication.
DNA, RNA, and Proteins
DNA codes for RNA, which guides protein synthesis
Central Dogma
After the discovery of DNA’s structure, scientists turned to investigating how DNA served as a genetic code for the synthesis of proteins.
Geneticists accept that the basic mechanism for reading and expressing genes is from DNA to RNA to protein.
This is referred to as the central dogma of biology: DNA codes for RNA, which guides the synthesis of proteins.
RNA
RNA is a nucleic acid similar to DNA, but with a different sugar, and with uracil instead of thymine.
Messenger RNA (mRNA)
: long strands of RNA that are formed complementary to one strand of DNA; direct synthesis of a specific protein
Ribosomal RNA (rRNA)
: associates with proteins to form ribosomes in the cytoplasm
Transfer RNA (tRNA)
: smaller segments of RNA that transport amino acids to the ribosome
Transcription
First step of the central dogma involves the synthesis of mRNA from DNA in a process called transcription.
The enzyme RNA polymerase regulates RNA synthesis by binding to the specific section where an mRNA will be synthesized.
RNA polymerase moves along the DNA strand in a 3’ to 5’ direction, synthesizing mRNA.
The Code
Scientists hypothesized that the instructions from protein synthesis were encoded in DNA.
Experiments during the 1960s demonstrated that the DNA code was a three-base code.
The three-base code in DNA or mRNA is called a codon.

Translation
After synthesis, mRNA moves from the nucleus into the cytoplasm, where it connects at the 5’ end to a ribosome.
The mRNA code is read and translated into a protein through a process called translation.
tRNA molecules act as the interpreters of the mRNA codon sequence.
The tRNA is activated by an enzyme that attaches a specific amino acid to the 3’ end.
The middle of the folded tRNA contains an anticodon, a complementary sequence to the mRNA codon.

Transcription and Translation
Click on the link below to watch a summary of transcription and translation processes.
http://www.teachertube.com/video/protein-synthesis-and-the-lean-mean-ribosome-machines-360142
DNA vs. RNA
The role of the Ribosome
Ribosomes provide a site for protein synthesis.
When mRNA leaves the nucleus, the two ribosomal subunits come together to hold the mRNA in place for translation.
The ribosome structure has grooves that hold serve as tRNA sites for amino acid attachment.

Gene Regulation and Mutation
Gene expression is regulated by the cell, and mutations can affect this expression.
Prokaryote Gene Regulation
Gene regulation is the ability of an organism to control which genes are transcribed in response to the environment.
An operon is a section of DNA that contains the genes for the proteins needed for a specific metabolic pathway.
An operon contains:
Operator
– on/off switch
Promoter
– where RNA polymerase binds
Regulatory gene
Genes
coding for proteins
The trp Operon
Tryptophan synthesis occurs in five steps controlled by the trp operon.
The trp operon is a repressible operon, because it is usually repressed or turned off.
The trp Operon
Click on the link below for a short video:
http://www.teachertube.com/video/trp-operon-217175
The lac Operon
When lactose is present, E. coli can synthesize an enzyme to use it as an energy source.
The lac operon is an inducible operon because an inducer binds to the repressor and inactivates it, allowing transcription to be turned on.

The lac Operon
Click on the link below to watch a short video:
http://www.teachertube.com/video/lac-operon-217174
Eukaryote Gene Regulation
Controlling transcription

Transcription factors ensure that a gene is used at the right time and that proteins are made in the right amounts.
Complexes that guide the binding of the RNA polymerase to a promoter
Regulatory proteins that help control the rate of transcription
The complex structure of eukaryotic DNA also regulates transcription.

Hox genes
Gene regulation is crucial during development and cell differentiation.
One group of genes that control cell differentiation is the homeobox (Hox) gene group.
Hox genes are transcribed at specific times in specific places on the genome, and control what body part will develop at a giving body location.

RNA interference
RNA interference (RNAi) can stop the mRNA from translating its message.
Single-stranded small interfering RNA and protein complexes bind to mRNA and stop translation.
Mutations
Types of mutations
A permanent change that occurs in a cell’s DNA is called a mutation.
Point mutation
: involve chemical change to just one base pair
Missense substitutions
: DNA codes for the wrong amino acid
Nonsense mutation
: Codon for amino acid becomes a stop codon
Insertion/deletion
: additions/ loss of a nucleotide to the DNA sequence
Cause “frameshifts”

Causes of mutation
Can occur spontaneously – DNA polymerase can attach the wrong nucleotide, but this is rare and usually corrected.
Certain chemicals and radiation called mutagens can damage DNA.
Chemicals can cause mispairing of base pairs, or themselves substitute for base pairs.
High-energy radiation can eject electrons from atoms within the DNA molecule, leaving behind unstable free radicals
Somatic cell mutations
are not passed on to the next generation.
Mutations that occur in sex cells
are passed on to the organism’s offspring and will be present in every cell of the offspring
Essential Questions
What are the different tools and processes used in genetic engineering?
How does genetic engineering manipulate recombinant DNA?
What are the similarities between selective breeding and genetic engineering?
How can genetic engineering and biotechnology be used to improve human life?
What are the components of the human genome?
How do forensic scientists use DNA fingerprinting?
How can information from the human genome be used to treat human diseases?
Researchers use genetic engineering to manipulate DNA.
DNA Tools
Genetic engineering can be used to increase/decrease the expression of specific genes in selected organisms.
An organism’s
genome
is the total DNA in the nucleus of each cell.
DNA tools can be used to manipulate DNA and to isolate genes from the rest of the genome.
Restriction enzymes
Restriction enzymes
are proteins that recognize and bind to specific DNA sequences and cleave the DNA within that sequence.
They are used as a defense mechanism by bacteria against viruses.
Scientists use restriction enzymes as powerful tools for isolating specific genes or regions of the genome

EcoRI
EcoRI is a restriction enzyme that specifically cuts DNA containing the sequence GAATTC.
Sticky ends are single stranded DNA sequences at the end of fragments.
Can be reattached to complementary strands
Restriction Enzymes
Gel electrophoresis
Gene cloning
To make a large quantity of recombinant plasmid DNA, bacterial cells are mixed with recombinant plasmid DNA.
Some of the bacterial cells take up the recombinant plasmid DNA through a process called
transformation
Bacteria that take up the plasmid make copies of the recombinant DNA during cell replication.
Large numbers of identical bacteria containing recombinant DNA can be produced through this process called cloning.
Recombinant DNA Technology
DNA fragments from different sources can be combined to make new DNA molecules.
The newly generated DNA molecule with DNA from different sources is called
recombinant DNA
.
Recombinant DNA is placed into bacterial cells for study via a carrier.
Common carriers include viruses and plasmids – small, circular, double-stranded DNA molecules that occur naturally in bacteria and yeasts.
DNA ligase
, a cellular repair enzyme, attaches the recombinant DNA to the plasmid.
DNA sequencing
Genetic engineering is technology that involves manipulating the DNA of one organism in order to insert the DNA of another organism.
The inserted outside DNA is known as exogenous DNA.
Genetically engineered organisms are used to:
Study the expression of a particular gene
Investigate cellular processes
Study the development of a certain disease
Select traits that might be beneficial to humans
Genetic Engineering
Click on the link below to watch a short video:
http://www.teachertube.com/video/restriction-enzymes-39228
An electric current is used to separate DNA fragments according to the size of the fragments in a process called
gel electrophoresis.
When an electric current is applied, the DNA fragments move toward the positive end of the gel.
The smaller fragments move farther faster than the larger ones.
The unique pattern created based on the size of the DNA fragment can be compared to known DNA fragments for identification.
Scientists study DNA sequences with DNA fragments, DNA polymerase, fluorescently labeled nucleotides, and gel electrophoresis.
Polymerase chain reaction
Once the sequence of a DNA fragment is known, a technique called the polymerase chain reaction (PCR) can be used to make millions of copies of a specific region of a DNA fragment.
PCR can copy or amplify a single DNA molecule numerous times for use in analysis.
PCR Technique
http://www.teachertube.com/video/dna-pcr-154513
Click on the following link to view the video:
Biotechnology
Biotechnology is the use of genetic engineering to find solutions to problems.
Organisms with genes from other organisms are called transgenic organisms.
Transgenic animals, plants, and bacteria are used for research, medicine, and agriculture.

Transgenic animals
Scientists produce most transgenic animals in laboratories for biological research.
They are used to:
Study diseases
Improve food supply
Improve human health
Be potential sources of organs for transplant

Transgenic plants
Frequently genetically engineered for resistance against insect or viral pests.
Other transgenic plants are designed to:
Reduce allergic reactions in humans
Contain increased vitamin and mineral content
Resist extreme weather
Produce vaccines or biodegradable plastics

Transgenic bacteria
Transgenic bacteria can:
Produce insulin and growth hormones
Slow the formation of ice on crops
Clean up oil spills
Decompose garbage
Genomes contain all of the information needed for an organism to grow and survive.
Human Genome Project
The goal of the
Human Genome Project (HGP)
was to determine the sequence of the approximately three billion nucleotides that make up human DNA and to identify all of the approximately 20,000-25,000 human genes.

Sequencing the genome
The 46 chromosomes were fragmented, combined with vectors, cloned, and sequenced using automated sequencing machines.
After sequencing, scientists observed that less than two percent of all the nucleotides code for all the proteins in the body.
The genome is filled with long stretches of noncoding sequences.
DNA fingerprinting
The long stretches of noncoding regions of DNA are unique to each individual.
DNA fingerprinting
involves separating the noncoding fragments to observe the distinct banding patterns that are unique to every individual
Identifying Genes
After sequencing the human genome, scientists began the process of identifying the genes and their functions.
For organisms without large noncoding regions, researchers have identified genes by scanning the sequence for Open Reading Frames (ORFs).
ORFs contain at least 100 codons that begin with a start codon and end with a stop codon.
Bioinformatics
The Human Genome Project and other sequencing projects produce enormous amounts of data.
Bioinformatics involves creating and maintaining databases of biological information.
Researchers analyze sequences, looking for genes and predicting the structures of newly discovered proteins.
DNA Microarrays
Gene expression can be analyzed using DNA microarrays, tiny microscope slides or silicon chips that are spotted with DNA fragments
Help researchers determine whether the expression of certain genes is caused by genetic factors or environmental factors.
The Genome and Genetic Disorders
Variations in the DNA sequence that occur when a single nucleotide in the genome is altered are called single nucleotide polymorphisms or SNPs.
For a variation to be considered an SNP, it must occur in at least one percent of the population.

The HapMap project
Regions of linked variations in a genome are known as haplotypes.
An international group of scientists is creating a catalog of common genetic variations that occur in humans.
Assembling the HapMap involves identifying groups of SNPs in a specific region of DNA.

Pharmacogenomics
The study of how genetic inheritance affects the body’s response to drugs is called
pharmacogenomics.
The benefits of pharmacogenomics include more accurate dosing of drugs that are safer and more specific to individuals.

Gene therapy
Gene therapy
is a technique aimed at correcting mutated genes that cause human diseases.
Scientists insert a normal gene into a chromosome to replace a dysfunctional gene.
Genomics and Proteomics
Genomics
is the study of an organism’s genome
Involves identifying genes and proteins produced by these genes.
Proteomics
is the large-scale study and cataloging of the structure and function of proteins.
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