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Transcript
2013 DESIGNER GENES
TRAINING GUIDE
by Karen L. Lancour
DISCLAIMER - This presentation was prepared using draft rules. There may be some changes in the final
copy of the rules. The rules which will be in your Coaches Manual and Student Manuals will be the
official rules.
 BE SURE TO CHECK THE 2013 EVENT RULES for EVENT PARAMETERS and TOPICS
FOR EACH COMPETITION LEVEL
TRAINING MATERIALS:
 Training Power Point presents an overview of material in the training handout
 Training Handout presents introductory topic content information for the event
 Sample Tournament has sample problems with key
 Event Supervisor Guide has event preparation tips, setup needs and scoring tips
 Internet Resource & Training Materials are available on the Science Olympiad website at
www.soinc.org under Event Information.
 A Biology-Earth Science CD as well as the Division B and Division C Test Packets are available
from SO store at www.soinc.org
Students will solve problems using their knowledge of Molecular Genetics, Biotechnology, and
Population Genetics. This event may be run as stations but it need not be. It is a very different event when
run as paper pencil. The best competition is still as stations using process skills and problem solving.
Every attempt should be made to avoid over-emphasis on a particular area.
1. At the various levels, areas to be tested include:
Regional & State
DNA structure & function
DNA Semi-conservative Replication
Gene expression (transcription and
translation
Control and detection of gene expression
Mutations
DNA Sequencing
Regional & State
Lac & Trp Operons
DNA
Fingerprinting/RFLP
DNA Sequencing
National (all topics)
Restriction mapping
Mitochondrial DNA
Plasmid selection and
isolation
PCR
Gene Therapy
Post- transcriptional modification
DNA Repair
Trinucleotide repeats
Epigenetics
Note: Regions or States may decide to cover all of the topics so check with your local tournament director for
specifics.
2. Process skills may include observations, inferences, predictions, data analysis, and calculations.
Note: It is a good idea to review the General Genetics Prinicples that are in the Heredity Event
in Division B – they are very relavent to Designer Genes covers.
1
MOLECULAR GENETICS
CENTRAL DOGMA OF MOLECULAR GENETICS
DNA ---- RNA --- PROTEIN SYNTHESIS
REPLICATION TRANSCRIPTION TRANSLATION
Central dogma of molecular genetics is DNA - RNA - Protein. Exception is in retroviruses
where genetic storage vehicle is RNA. It then makes a DNA which replicates to form double
stranded
DNA and continues through dogma.
Exceptions among viruses – RNA to DNA (retroviruses)
DNA Structure
DNA structure – double helix with sugar (deoxyribose), phosphate and nitrogen bases (Adenine,
Thymine, Guanine, and Cytosine) Pairing – A with T and G with C
Nucleotide - basic unit of sugar, phosphate and nitrogen base - 4 kinds of nucleotides because of the
types of bases
2
4
DNA Replication
DNA replication is semi-conservative and occurs in the nucleus.
Events that occur:




DNA polymerase is the key enzyme
DNA uncoils and splits
template strand is read 3’ to 5’
new complementary strand must add new nucleotides to the 3’ end – leading strand
(continuous) while lagging strand is fragments (Okazaki fragments) latter attached with
the enzyme ligase
DNA Repair Genes encode proteins that correct mistakes in DNA caused by incorrect copying
during replication and environmental factors such as by-products of metabolism, exposure to
ultraviolet light or mutagens. The DNA repair process must operate constantly to correct any
damage to the DNA as soon as it occurs.
3
ENZYMES INVOLVED IN REPLICATION
The replication fork is the unwound helix, with each strand being synthesized into a new double
helix

Topoisomerase is responsible for initiation of the unwinding of the DNA.

Helicase accomplishes unwinding of the original double strand, once supercoiling has been
eliminated by the topoisomerase.

DNA polymerase (III) proceeds along a single-stranded molecule of DNA, recruiting free
dNTP's (deoxy-nucleotide-triphosphates) to hydrogen bond with their appropriate
complementary dNTP on the single strand (A with T and G with C), and to form a covalent
phosphodiester bond with the previous nucleotide of the same strand.
DNA polymerases cannot start synthesizing de novo on a bare single strand. It needs a primer
with a 3'OH group onto which it can attach a dNTP.
DNA polymerase also has proofreading activities, so that it can make sure that it inserted the
right base, and nuclease (excision of nucleotides) activities so that it can cut away any
mistakes it might have made.

Primase attaches a small RNA primer to the single-stranded DNA to act as a
substitute 3'OH for DNA polymerase to begin synthesizing from. This RNA primer is
eventually removed and the gap is filled in by DNA polymerase (I).

Ligase can catalyze the formation of a phosphodiester bond given an unattached but adjacent
3'OH and 5'phosphate. This can fill in the unattached gap left when the RNA primer is
removed and filled in.

Single-stranded binding proteins are important to maintain the stability of the replication fork.
Single-stranded DNA is very labile, or unstable, so these proteins bind to it while it remains
single stranded and keep it from being degraded.
4
Nuclear vs Cytoplasmic DNA in Eukaryotic Cells
 Nuclear DNA – in chromosomes within the nucleus of the cell
 Cytoplasmic DNA – in chloroplasts and mitochondria
• Chloroplast DNA (cpDNA)
• Mitochondrial DNA (mtDNA)
 Features:
• Maternal inheritance
• Resemble prokaryotic DNA
• Slow accumulation of mutations
•
Differences between RNA & DNA
 RNA is single strand - DNA is double strand
 RNA has Ribose – DNA has Deoxyribose
 RNA has Uracil – DNA has Thymine
Gene Expression
There are two processes with utilize the DNA template code to ultimately produce proteins:
 Transcription – DNA is template for making RNA (in nucleus) There are 3 types of RNA.
 Translation (protein synthesis) - in cytoplasm at the ribosome. M-RNA has blueprint, T-RNA
transfers amino acids, and Ribosome (R-RNA) allows T-RNA to attach to M-RNA at appropriate
site.
 many factors control gene expression including:
o
factors affecting DNA structure,
o
gene expression,
o
factors affecting assembly of proteins after
o
translation,
o
hormones,
o
environmental factors as viruses.
Types of RNA
Kinds of RNA – three kinds of RNA are produced in the nucleus using DNA coding strands



Messenger RNA (m-RNA) – carries genetic code from DNA into cytoplasm
Transfer RNA (t-RNA) – brings the amino acids for building of protein to be attached according
to the genetic code of the M-RNA
Ribosomal RNA (r-RNA) – make up the ribosome and reads the code of M-RNA and allow TRNA to attach and connect amino acids
5
Transcription
Transcription - production of RNA in the nucleus using a DNA segment as a template and RNA
polymerase as the key enzyme.
Post-transcription Modifications
RNA’s are modified in eukaryotes before leaving the nucleus.
 PreM-RNA has exons (coding segments) and introns (noncoding segments between exons)
 introns (the noncoding segments) are removed
 a cap is added to the 5’ end
 a poly A tail is added to the 3’ end before it leaves the nucleus
6
Universal Code (Codon = Amino Acid)






Each three base codon on the messenger RNA (m-RNA) is a code for an amino acid
There are 64 possible three base codons – 61 are codes for one of the 20 amino acids
The three remaining codons are termed stop codons because the signal the end of a peptide segment
Notice that many of the amino acids have more than one codon
A three base code on the DNA produces the mRNA codon
The three base code on the t RNA is termed an anticodon because it will bond to a m-RNA codon
during translation or protein synthesis
7
Translation (Protein Synthesis)
Translation – genetic code used to form amino acid sequence using M-RNA, T-RNA, and R-RNA
(ribosomes) occurs in the cytoplasm at the ribosome. Many key enzymes (proteins) are involved.
8
Translation (Protein Synthesis)
The steps of translation:

Initiation: a mRNA enters the cytoplasm and becomes associated with ribosomes (rRNA +
proteins) and tRNAs, each carrying a specific amino acid, pair up with the mRNA codons inside
the ribosomes. The base pairing (A-U, G-C) between mRNA codons and tRNA anticodons
determines the order of amino acids in a protein.

Elongation: involves the addition of amino acids one-by-one: As the ribosome moves along the
mRNA, each tRNA transfers its amino acid to the growing protein chain, producing the protein

Termination: when the ribosomes hits a stop codon - UAA, UGA, or UAG - – no tRNA with its
amino acid can be added so the ribosome falls apart and the process ends. The same mRNA may be
used hundreds of times during translation by many ribosomes before it is degraded (broken down)
by the cell.
A
close up showing the M-RNA (with its codon) and T-RNA (with it anticodon as well as the Amino
Acid) attaching at the P and A sites on the Ribosome.
Control of Gene Expression








Transcriptional Control
Post transcriptional Control – assembling proteins
Cell differentiation and specialization
Turning genes “on” and “off”
Chemical Signals – Hormones
Chemical Modifications
Relocation of DNA – transposons
Abnormal Expression of Genes
9
Mutations
 Gene – section of DNA with carries the blueprint for making a peptide strand or RNA.
 Mutation – changes in genetic code (DNA blueprint) of genes or chromosomes and causes changes in
expression in the for making protein or RNA
 Gene mutation
 Chromosomal mutation
 Agents causing mutations – radiation, chemicals, excess heat , viruses
Genetic Disorders











Nondisjunction – extra or missing chromosomes as Down’s Syndrome
Trinucleotide repeats – triplet nucleotides
repeated too often as Huntington’s
Defective genes – does not produce correct protein as sickle cell anemia (A & T traded places)
Causes of mutations – chemicals, radiation, temperature, viruses
Genetic disorders and their causes as nondisjunction (Down’s syndrome), trinucleotide
repeats (fragile X and Huntington’s), defective genes (sickle cell anemia, hemophilia)
Trinucleotide repeats – sequences of 3 nucleotides is repeated, often several times in a gene,
when too many repeats are formed – cause genetic disorders
Nondisjunction – chromatids do not separate properly during meiosis. Individual formed
from such gametes have extra or missing chromosomes.
Human genetic disorders – can be dominant, recessive, sex-linked, epistatic, variable
expressed
Crossover frequency – during meiosis, pieces trade places – determining frequency
Mitochondrial Inheritance – genetic make-up of mitochondria, genetic code and patterns
transmitted through mother.





The inheritance of a trait encoded in the
mitochondrial genome
Mitochondrial DNA or mtDNA is
inherited from the mother
The mtDNA is circular and resembles
prokaryotic DNA
The mitochondria are responsible for
energy production
Mitochondria can reproduce independent
of the rest of the cell – an advantage in
energy production

Persons with a mitochondrial disease may be
male or female but they are always related in
the maternal line and no male with the
disease can transmit it to his children

Mitochondrial myopathies are a group of neuromuscular diseases caused by damage to the
mitochondria-small, energy-producing structures that serve as the cells' "power plants."
10
Lac & Trp Operons - examples of prokaryotic gene regulation
 Many of the prokaryotic genes as in E.coli are expressed or are always turned "on".
 Others are active only when their products are needed by the cell, so their expression must be
regulated.
 Examples of Operons in E. coli
o The genes for the five
enzymes in the Trp synthesis
pathway are clustered on the
same chromosome in what is
called the Trp Operon - If
the amino acid tryptophan
(Trp) is added to a culture of
E coli , the bacteria soon stop
producing the five enzymes
needed to synthesize Trp
from intermediates produced
during the respiration of
glucos so the presence of the
products of enzyme action
represses enzyme synthesis
This is a repressable operon
where genes are expressed in
the absense of a substance and the presense of the substance shuts off the gene
o The genes that code for the enzymes
needed for lactose catabolism are
clustered on the same chromosome in
what is called the Lac Operon –
prokaryotics as E. coli have a mechanism
for metabolizing lactose – the sugar used
for energy. Three proteins or enzymes are
needed in lactose metabolism and they are
encoded in a single expressible unit of
DNA called the lac operon The E. coli
only express the genes and make these
enzymes when lactose is available to be
metabolized. This is an inducible operon
where genes are expressed in the presence
of a substance
11
Biotechnology – technology to manipulate DNA – techniques often called genetic engineering or
Recombinant DNA Technology.






Technology used to manipulate DNA
Procedures often called genetic engineering
Recombinant DNA - DNA from two sources
Transgenic individuals have DNA from another organism
Often involve putting genes into viruses or bacteria.
Vectors are the pieces of DNA used to transfer genes into a host cell – often plasmids of bacteria
Overview of Biotechnology
12
Basic Tools of DNA Technology


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
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
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

Identifying desired DNA
Cutting DNA with Restriction Enzymes
Inserting DNA into Vector as Plasmid
Connecting DNA pieces with Ligase
Inserting Vector into Host Cell as bacterium
Cloning desired DNA and Vectors
Storing clones in DNA Libraries
Identifying cloned genes with Radioactive Probes
Analyzing DNA by cutting fragments and separating by Electrophoresis
DNA Analysis Technologies
 identifying – recognizing desired DNA fragment or plasmid using radioactive probes
 cutting DNA - using desired restriction enzymes or “ enzymatic sissors”
 making hybrids of DNA using Hybridization techniques
 cloning DNA – using other cells or in a test tube as with PCR – Polymerase Chain Reaction – clones
DNA segments in a test tube quickly and inexpensively. May use very small amounts of DNA to
clone
 storing DNA in DNA libraries of plasmids or bacteriophages of genome DNA or cDNA.
 separating DNA segments with electrophosesis
 transferring DNA using blotting
 imaging DNA with autoradiography
 analyzing DNA by sequencing or determining the nucleotide sequence of a gene, microassays
analyze gene function and expression, DNA fingerprinting techniques as RFLP or restriction
fragment length polymorphism, VNTRs or Variable Number Tandem Repeats, STRs or Short
Tandam Repeats, Ribosomal DNA Analysis, or Y-chromosome Analysis
Basic Terminology
 Recombinant DNA – DNA from two different sources combined. Often involve putting genes
 into viruses or bacteria using a vector.
 Inserting a gene into a bacterium - Organism provides the desired piece of DNA which is spliced
into a piece of DNA used to transfer the genes or vector which is inserted to a Host cell (often a
bacterium)
 Plasmids– in bacteria, circular DNA serve as vectors. Easily taken up by bacterial cells. It is more
difficult to insert vector into Eukaryotic cells.
 Transgenic organisms have DNA from another organism
 Restriction enzymes - enzymes to cut DNA at a particular spot and DNA ligase enzymes reattach
ends.
 Hybridization – process of putting pieces of DNA together.
 Chromosome mapping – determining the location of genes on a chromosome and making a map
 of restriction sites as Retriction Maps.
13
Basic Tools

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
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

Gene selection & isolation from Donor
Vector - Plasmid selection & isolation
Restriction enzyme to cut
piece
Putting pieces together
(hybridization)
DNA ligase to reattach pieces
Insert into Host bacteria
Clone the bacteria
14
cDNA – complementary DNA
 cDNA- Eukaryotic genes contain introns but bacteria do not contain the necessary enzymes to remove
introns
 Eukaryotic genes that are inserted into bacteria must be inserted without introns.
 Use reverse transcriptase (from retroviruses) and modified M-RNA to produce cDNA with introns
already removed
Techniques




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

Storing clones in DNA Libraries
Cloning within cells and with PCR
Identifying cloned genes with Radioactive Probes
Analyzing DNA by cutting fragments and separating by Electrophoresis/nucleic acid
hybridization/DNA probes
Transferring DNA from gel by Blotting
Imaging with autoradiography
DNA Sequencing to determine exact sequence
Microassays to analyze gene function
DNA Libraries
 Genomic – normal DNA
 cDNA – modified to remove
introns
 Fragments stored
 Stored in plasmids or
bacteriophages
15
Polymerase Chain Reaction (PCR)
 Technique for quickly making an unlimited number of copies of any piece of DNA
 Sometimes called "molecular photocopying
16
Radioactive Probes
 short, radioactive strands of DNA
 will pair up with complementing strands of DNA
 fragments that contain the labeled pieces will show up on an x-ray film
Electrophoresis
A process in which molecules (such as proteins, DNA, or RNA
fragments) can be separated according to size and electrical
charge by applying an electric current to them.
DNA Hybridization
 Base pairing of two single
strands of DNA or RNA.
 Can be DNA-DNA, DNA-RNA
 Can be a radioactive probe
17
DNA Analysis
DNA Sequencing
 Determine the exact nucleotide
sequence
 Columns for A,T,C, and G
 Read rows from top to bottom
18
Restriction Mapping
 Description of restriction enzyme cleavage sites within a piece of DNA
 Use of different restriction enzymes to analyze and generate a physical map of genomes, genes, or
other segments of DNA
DNA Microassay
 studying how large numbers of genes interact with each
other
 precisely apply tiny droplets containing functional DNA to
glass slides
 attach fluorescent labels to DNA from the cell they are
studying.
 labeled probes are allowed to bind to complementary DNA
strands on the slides
 slides are put into a scanning microscope that can measure
the brightness of each fluorescent dot
 brightness reveals how much of a specific DNA fragment is
present, an indicator of how active it is.
19
DNA Fingerprinting Techniques






RFLP – Restriction Fragment Length Polymorphism (original)
PCR – Polymerase Chain Reaction
VNTRs – Variable Number Tandem Repeats
STRs - Short Tandem Repeats
Ribosomal DNA analysis
Y-chromosome analysis
Restriction Fragment Length Polymorphism – RFLP







The DNA of an organism is cut up into fragments using restriction enzymes.
A large number of short fragments of DNA will be produced. (RFLP’s)
Electrophoresis is a technique used to separate the DNA fragments according to
their size.
Uses- identification of diseased genes including oncogenes, identification of
viral infections, determining family relationships among individuals, and
identifying tissue found at a crime scene.
Genetic variations at the site where a restriction enzyme cuts a piece of DNA.
Such variations affect the size of the resulting fragments.
These sequences can be used as markers on physical maps and linkage maps.
20
VARIABLE NUMBER TANDAM REPEATS (VNTR’s)
 Short nucleotidesequences
 Organized in clustersof tandem repeats
 VNTR = 14-100 base pairs
 SNR = 2- 10 base pairs
21
Applications of Biotechnology Techniques







Human Genome Project - entire gene make up of humans
Diagnosis of Disease – PCR & DNA probes
Human Gene Therapy
Vaccines & Pharmaceutical Products
Forensics – DNA Fingerprints (RFLP & VNTR)
Environmental – Recycling & detoxification
Agricultural – transgenic organisms
Gene Therapy - changing the expression of a person’s genes - body (somatic) or germ cells
done invitro or exvitro.
Bioethics
Major concerns concerning safety and ethics of recombinant DNA technology.


Potential Hazards vs. Potential Gains
Concerns:
o
genetically modified foods
o
genetically engineering microbes
o
cloning whole organisms
o
embryonic stem cell research
o
gene therapy
o
genetic testing
o
bioterrorism
Epigenetics is the study of heritable changes in gene activity that occur without a change in the sequence
of the genetic material. Epigenetics literally means ‘in addition to genetics’.
 Epigenetic factors can regulate the amount of gene activity, influencing the growth and appearance of
an organism
 There are several epigenetic ways in which gene activity can be prevented or controlled, including
o modification of histone proteins
o DNA methylation
o RNA interference
 For any of these methods of gene regulation, the absence of the protein product of the gene causes a
change in the function or development of the cell

malfunctions in epigenetic control of gene activity have been implicated in cancer, cardiovascular
disease and several inherited genetic conditions
22
SAMPLE STATION PROBLEMS
 DNA structure and Replication
 Production of RNA from a coding strand of DNA - & post transcription modification of M-RNA
 Protein Synthesis Analysis (Replication and Transcription)
 Control and Detection of Gene Expression
 Chromosome map units problems
 Cross over frequencies
 DNA sequencing
 Restriction enzyme analysis – Making a restriction map of a plasmid or DNA segment.
 Interpreting data from DNA fingerprinting studies
 Forensic Uses of Biotechnology
 Interpreting DNA analysis data + comparing RFLP and PCR forensic testing and analysis
 Analyzing blood chemistry, blood typing and blood cell genetic information
 Analyzing and interpreting chromosome maps
 Understanding and interpreting the uses of restriction enzymes and radioactive probes
23