American College of California Biology Lectures Worksheet

  

due december 12 – 11am EASTERN TIME

i have attached all lecture slides for each lecture to make it easier for u 🙂

Homework 10 part 1-3 – Lecture 25-27
Lecture 25
1. The following is a physical map of a region you are mapping for RFLP analysis:
The numbered vertical lines represent restriction sites recognized by SmaI. Sites 2 and
3 are polymorphic, the others are not. You cut the DNA with SmaI, electrophorese the
fragments, and Southern blot them to a membrane. You have a choice of two probes
that recognize the DNA regions shown above: probe A, short; probe B, long.
(a) Explain which probe you would use for analysis and why the other choice would be
unsuitable.
(b) Give the sizes of bands you will detect in individuals homozygous for the following
genotypes with respect to sites 2 and 3.
Haplotype
A
B
C
D
Site 2
Present
Present
Absent
Absent
Site 3
Present
Absent
Present
Absent
2. A cigarette butt found at the scene of
a violent crime is found to have a
sufficient number of epithelial cells
stuck to the paper for the DNA to be
extracted and typed. Shown below
are the results of typing for three
probes (locus 1, locus 2, and locus
3) of the evidence (X) and 4
suspects (A through D). a) Which of
the suspects can be excluded? b)
Which cannot be excluded? c) Can
you identify the criminal? Explain
your reasoning. d) Why are there two
bands for each individual?
3. Based on the DNA fingerprinting
(minisatellite analysis with a multilocus
probe) results shown in the figure
below, is Mr. X or Mr. Y the child’s
father? Explain your answer.
4. a) Complete the incomplete diagrams below to show the key structural difference
between an NTP, dNTP, and ddNTP (e.g., CTP, dCTP, ddCTP). Write your answers
below the structures in the text section.
(b) What do “d” and “dd” stand for?
(c) Explain why ddNTPs are called “chain terminators” in DNA sequencing reactions.
NTP – 3’C=
dNTP – 3’C=
ddNTP – 3’C=
2’C=
2’C=
2’C=
Lecture 26
1. How has the human genome project facilitated identification of disease genes?
2. After sequencing the entire genome of a novel organism by the whole-genome
shotgun approach, you use a computer program to search for start/stop signals for
translation and homology searches based on similarity to cDNAs in the database.
You predict approximately 20,000 genes. Is this an accurate representation of the
total number of genes in the organism? Why or why not?
3. Design a genomics experiment to discover new genes involved in the process of cell
cycle regulation. Clearly describe the experiment, state key reagents and expected
type of results.
4. You perform a co-immunoprecipitation experiment of your favorite mouse protein
YMFP), perform mass spectrometry to identify the interacting factor and find that
YFMP associates with a protein that has only been given a number, CG1024, not a
name. Describe some initial analysis that you would do to find out about the
interacting factor CG1024.
5. You determined that your interacting factor has a zinc finger motif. What is the likely
role for this protein? Design genomic and/or proteomic experiments to test the
putative function of your protein. Clearly describe the experiment, state key reagents
and expected type of results.
6. Compare and contrast the techniques used in, and information obtained from,
transcriptomics and proteomics.
Lecture 27
1. After SNP analysis of a region of the Apolipoprotein E gene, you determine that a
patient carries the apoE4 allele. This allele is linked with Alzheimer’s disease.
Counsel the patient regarding their prognosis.
2. Predict the effects that a SNP would have it were located in an
a) intron
b) exon
c) intragenic region
3. In a study that involved 100 Caucasian males, you find a polymorphism in a gene for
a neurotransmitter receptor. The “long allele” is linked with reckless behavior. What
conclusions, if any, can you draw about the results of this study? Should you hold a
press conference to announce the discovery of the “reckless” gene?
Fundamental
Molecular Biology
Second Edition
Lisabeth A. Allison
Chapter 16
Genome Analysis:
DNA Typing, Genomics, and Beyond
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Cover photo: Julie Newdoll/www.brushwithscience.com “Dawn of the
Double Helix”, oil and mixed media on canvas, © 2003
Learning Objectives
• Methods of DNA typing
•
•
•
•
RFLP
Minisatellite
STR
Mitochondrial DNA
• DNA sequencing approaches
• Sanger sequencing
• next generation sequencing
• Levels of genome analysis range from
personal identification to comparative
analysis of entire genomes.
16.2 DNA typing
• One of the most reliable and conclusive
methods available for identification of an
individual.
• Technique developed by Alec Jeffrey’s
and coworkers in 1985.
• First called “DNA fingerprinting,” now
called “DNA typing.”
Applications of DNA typing
• Establish paternity and other family
relationships.
• Identify potential suspects whose DNA may
match evidence left at a crime scene.
• Exonerate persons wrongly accused of crimes.
• Match organ donors with recipients in
transplant programs.
• Identify catastrophe victims.
• Detect bacteria and other organisms that may
pollute air, water, soil, and food.
• Determine whether a clone is genetically
identical to the donor nucleus.
• Trace the source of different marijuana plants.
• Identify endangered and protected species as
an aid to wildlife officials.
DNA polymorphisms: the basis of
DNA typing
•
•
•
•
•
•
•
•
Only about 0.1% of the human genome differs from one person to
another.
With the exception of the human leukocyte antigen (HLA) region, genetic
variation is relatively limited in coding DNA.
Less than 40% of the human genome is comprised of genes and generelated sequences.
Intergenic DNA consists of unique or low copy number sequences and
moderately to highly repetitive sequences.
The majority of DNA typing systems used in forensic casework are based
on genetic loci with minisatellites or short tandem repeats (STRs).
Analyze multiple variable regions, called polymorphic markers.
The power of DNA evidence lies in statistics.
Aim to calculate the probability that only one person in a quadrillion (1015)
could have the same profile of markers.
Sequence Type in the Human
Genome
8.6 RFLP analysis
Restriction fragment length
polymorphism (RFLP)
• The existence of alternative alleles associated
with restriction fragments that differ in size from
each other.
• Variable regions do not necessarily occur in
genes.
• Function of most RFLPs in the human genome
is unknown.
– Exception: sickle cell anemia RLFP
Diagnosis of sickle cell anemia by restriction
fragment length polymorphism (RFLP)
and Southern blot
• A point mutation in the -globin gene has
destroyed the recognition site of the restriction
endonuclease MstII.
• Affected individuals: larger restriction fragment
on a Southern blot
• Normal individuals: shorter restriction fragment
Restriction fragment length
polymorphism (RFLP)
RFLPs can serve as markers of
genetic disease
• A RFLP that is close to a disease gene tends to
stay with that gene during crossing-over
(recombination) during meiosis.
• Linkage: the likelihood of having one marker
transmitted with another through meiosis.
• When a PCR assay for typing a particular locus
is developed, it is generally preferable to RFLP
analysis.
A variety of DNA technologies are used in
forensic investigations:
•
•
•
•
•
•
Minisatellite analysis
PCR-based analysis
STR analysis
Mitochondrial DNA analysis
Y chromosome analysis
Random amplified polymorphic DNA
(RAPD) analysis
Minisatellite analysis
• Minisatellites are a special class of RFLP
in which the variable lengths of the DNA
fragments result from a change in the
number, not the base sequence, of
minisatellite repeats.
• Also known as variable number tandem
repeats (VNTRs).
Minisatellite analysis with a single-locus
probe
• A single-locus probe allows the detection
of a single minisatellite DNA locus on one
chromosome.
• To increase the sensitivity, 3-5 singlelocus probes are mixed in a single-locus
“cocktail.”
Minisatellite
analysis
Classic “DNA fingerprinting:”
minisatellite analysis with
a multilocus probe
• Unique biological identifier for each
individual.
• Essentially constant for an individual,
irrespective of the source of DNA.
• Simple Mendelian pattern of inheritance.
• Requires relatively large amounts of DNA.
• Does not work well with degraded
samples.
Short tandem repeat analysis
• Currently the most widely used DNA
typing procedure in forensic genetics.
• The variability in STRs mainly occurs by
slippage during DNA replication, rather
than by unequal crossing-over.
Polymerase chain reaction-based
analysis
• Sufficient DNA can be collected from
saliva on a postage stamp or bones from
skeletons.
• Even highly degraded DNA can be
amplified, as long as the target sequence
is intact.
Multiplex analysis of STRs
• Simultaneous amplification of many
targets of interest in one reaction by
using more than one pair of primers.
• The FBI uses a standard set of 13
specific STR regions for CODIS (The
Combined DNA Index System).
Short tandem repeats (STR) Microsatellites
Example:
• 15 different STRs and a gender-specific
marker amplified by PCR.
• One primer in each pair is labeled with a
fluorescent tag for 4 color detection.
• Detect PCR amplification products using an
automated sequencer.
• Separated by size and detected by color after
laser-induced excitation.
Tools to speed the process of
mapping STRs
Mitochondrial DNA analysis
• Every cell has hundreds of mitochondria with
several hundred mtDNA molecules.
• Older biological samples (e.g. strands of hair,
solid bone, or teeth) often lack usable nuclear
DNA but have abundant mtDNA.
• mtDNA has been successfully isolated from
fossil bones.
• Analysis by PCR amplification and direct
sequencing of two highly variable regions in
the D loop region.
• Can only identify a person’s maternal lineage.
mtDNA
Y chromosome analysis
• Y chromosome-specific STRs.
• Paternity testing of male offspring
• Analyzing biological evidence in criminal
casework involving multiple male
contributors.
Randomly amplified polymorphic DNA
(RAPD) analysis
• No knowledge of an organism’s DNA
sequence is required.
• PCR primers consist of random sequences.
• e.g. The case of the Palo Verde tree seed
pods.
• e.g. Differentiation between Bacillus species.
8.7 DNA sequencing
DNA sequencing is the ultimate
characterization of a cloned gene.
• Manual sequencing by the Sanger “dideoxy”
DNA method.
• Automated DNA sequencing.
• Next-generation sequencing.
Manual sequencing by the Sanger
“dideoxy” DNA method
A DNA synthesis reaction:
• DNA polymerase (T7 DNA polymerase called
“Sequenase”)
• DNA template
• Free 3′-OH to get the polymerase started
• dNTPs
DNA sequencing involves
DNA synthesis
DNA synthesis requires a free
3’ OH
Sanger sequencing uses
dideoxy nucleotides which
are chain terminators
Sanger Sequencing
Sequencing animation
• https://www.dnalc.org/resources/animati
ons/sangerseq.html
Sanger
Sequencing
Automated DNA sequencing
• Developed by Leroy Hood and Lloyd Smith in
1986.
• Each ddNTP terminator is tagged with a different
color of fluorophore.
• DNA samples loaded in a capillary array migrate
through a gel matrix by size, from smallest to
largest.
Automated DNA sequencing
• When DNA fragments reach the detection
window, a laser beam excites the fluorophores
causing them to fluoresce.
• An electropherogram―a graph of fluorescence
intensity versus time―is converted to the DNA
sequence by computer software.
Automated
Sequencing
Next-generation sequencing
• The sequencing of spatially separated, clonally
amplified DNA templates in a massive array all
at the same time.
• DNA sequences in the range of hundreds of
megabases to gigabases can be rapidly
obtained.
454 pyrosequencing
• Template DNA is prepared by emulsion PCR.
• The template DNA is immobilized on a bead in a
well in the sequencing machine.
• Solutions of A,C,G, and T nucleotides are
sequentially added and removed from the
reaction.
• The enzyme luciferase is used to generate light.
• Light is only produced when the nucleotide
solution complements the first unpaired base of
the template.
Next Generation
Sequencing
Fundamental
Molecular Biology
Second Edition
Lisabeth A. Allison
Chapter 16
Genome Analysis:
DNA Typing, Genomics, and Beyond
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Cover photo: Julie Newdoll/www.brushwithscience.com “Dawn of the
Double Helix”, oil and mixed media on canvas, © 2003
Learning Objectives
• Incentives and challenges of the human
genome project
• Whole genome sequencing approaches
• Map based clone contig
• Shot-gun
• Genome Annotation
• Importance of comparative analysis
• BLAST
16.4 Whole genome sequencing
Organization of the human genome
• Less than 40% of the human genome is
comprised of genes and gene-related
sequences.
• Intergenic DNA consists of unique or low
copy number sequences and moderately
to highly repetitive sequences.
Repetitive DNA sequences are divided
into two major classes
• Interspersed elements
• Tandem repetitive elements
Interspersed elements are primarily
transposable elements
Genome-wide repeats that are primarily
degenerate copies of transposable
elements
• Short interspersed nuclear elements (SINEs)
• Long interspersed nuclear elements (LINEs)
Tandem repetitive sequences are
arranged in arrays with variable
numbers of repeats
Three subdivisions based on length
• Satellite DNA
• Minisatellites
• Short tandem repeats (STRs)
Sequence Type in the Human
Genome
Incentive of Human Genome
Project
•
•
•
•
Revolutionize human genetic medicine
Mutation rates
Cancer cells
Other human diseases
Challenges
• Scale
• Cost and time
• Amount of repetitive sequence
• Ethical concerns
Two main genome sequencing methods
• Clone by clone genome assembly approach:
– Used by the publicly funded international
sequencing consortium for the human
genome.
• Whole-genome shotgun approach:
– Used by the privately funded Celera
Genomics Corporation for the human
genome.
Clone by clone genome
assembly approach
• Restriction fragments of ~150 kb are cloned
into BAC vectors.
• A physical map of the genome is produced.
• The BAC clones are broken up into smaller
fragments, subcloned, and sequenced.
• This places the sequences in order so they can
be pieced together.
• Time consuming, but precise.
Whole-genome
shotgun approach
• Plasmid clones with 2-10 kb inserts are
prepared directly from fragmented genomic
DNA.
• Clones are randomly selected for sequencing.
• Sequence is reassembled in order with the aid
of a supercomputer.
• More rapid, but often results in gaps in the
sequence.
Map-based, Clone contig
Assemble first, then sequence
IHGSC
Shot-gun
Sequence first, then assemble
Celera Genomics
Rough drafts versus finished
sequences
• “Rough draft” of the human genome
reported in 2001 by the publicly and
privately funded groups.
• “Finished” sequence reported in 2004.
• More accurate and complete, but still
contains some gaps.
Sequence Finishing
We know the sequence, now
what?
Genome Annotation
• Prediction of transcript and protein
sequence
• Prediction of function for each predicted
protein
What is a gene and how many are
there in the human genome?
Three essential features of a gene:
• Expression of a product.
• Requirement that it be functional.
• Inclusion of both coding and regulatory
regions.
Gene Identification
Search for genes by looking
for gene landmarks
Search for genes by looking
for gene landmarks
Local Alignment –
BLAST
Basic Local
Alignment Search
Tool
Comparative
Analysis
Genome Annotation
• Prediction of transcript and protein
sequence
• Prediction of function for each predicted
protein
GO – Gene Ontology
• Functional classification scheme
• Molecular function, biological role, cellular
location
• Data from fly, yeast, mouse
• Comparison against databases that list
genes with known function
• The Encyclopedia of DNA Elements
(ENCODE) Project aims to identify all
functional elements in the human genome.
• This includes protein-coding genes, nonprotein-coding genes, transcriptional
regulatory elements, and sequences that
mediate chromosome structure and
dynamics.
What’s next?
• Knowing fly, worm, yeast sequence enhances
usefulness as a model organism
• Reverse genetics
• Whole genome approaches
• Lead to understanding complex regulatory
networks
16.3 Genomics, proteomics,
and beyond
• Whereas gene discovery once drove
DNA sequencing, now the sequencing of
entire genomes drives gene discovery.
What is bioinformatics?
• Area of computer science devoted to
collecting, organizing, and analyzing
DNA and protein sequences and all the
data being generated by genomics and
proteomics labs.
Tools of bioinformatics:
•
•
•
•
Locate and align sequences.
Assemble consensus sequences.
Analyze properties of proteins.
Analyze sequence patterns to locate
restriction sites, promoters, DNA binding
domains, etc.
• Phylogenetic analysis.
• Basic local alignment search tool
(BLAST).
• The most commonly used genome tool.
• Example: Search for all the predicted
protein sequences that are related to a
“query sequence.”
Genomics
• The comprehensive study of whole sets
of genes and their interactions rather
than single genes.
• Comparative analysis of genomes based
on the availability of complete genome
sequences.
Proteomics
• The comprehensive study of the full set
of proteins encoded by a genome—the
“proteome.”
• Protein biochemistry on a “highthroughput” scale.
The age of “omics” and
systems biology
• A whole set of related terms coined to describe
the comparative study of databases.
– e.g., transcriptomics, metabolomics,
kinomics, glycomics, lipidomics
• Interactomics: the study of macromolecular
machines, mapping protein-protein interactions
throughout a cell.
• Systems biology aims to make sense of all
the data arising from the study of
biomolecular networks.
• Uses both experimental and computational
approaches to model these interactions.
• “Attempts to piece together everything.”
16.5 High-throughput analysis
of gene function
Learning Objectives
•
•
•
•
•
•
Microarray analysis
RNA-seq
ChIP-chip, ChIP-seq
Yeast knock out collection
Yeast two hybrid and interaction maps
Co-immunoprecipitation
DNA microarrays
• Analysis of the transcriptional activity of
thousands of genes simultaneously.
• Compare transcription programs of cells
or organisms during specific
physiological responses, developmental
processes, or disease states.
Microarray
RNA-seq
http://cmb.molgen.mpg.de/2ndGenerationSequencing/Solas/RNA-seq.html
RNA-seq
http://www.nature.com/nbt/journal/v28/n5/fig_tab/nbt0510-421_F1.html
ChIP
ChIP-Seq examples
Figure 1
Molecular Cell 2017 68, 773-785.e6DOI: (10.1016/j.molcel.2017.10.013)
Copyright © 2017 Elsevier Inc. Terms and Conditions
Mass spectrometry
Two popular strategies:
• Peptide mass fingerprinting using
MALDI-TOF.
• Shotgun proteomics using MS/MS
Peptide mass fingerprinting using
MALDI-TOF
• Analysis of a single isolated protein.
• MALDI-TOF (Matrix Assisted Laser
Desorption/Ionization-Time of Flight)
mass spectrometry.
• Time of flight is inversely proportional to mass
and directly proportional to charge.
• Measurement of the number of ions at each
m/z value (mass to charge ratio).
• Computer database: identify protein from which
peptides originated.
Shotgun proteomics using MS/MS
• “Interrogation” of an entire proteome.
• Tandem mass spectrometry (MS/MS).
• The process produces a collection of peptide
ion fragments that differ in mass by a single
amino acid.
• Measurement of mass to charge (m/z) ratios of
the fragments allows the amino acid sequence
to be read.
Peptide Mass Fingerprinting
Identification of interacting proteins
and/or complexes
The nucleolar proteome
• Analysis of the nucleolar proteome by
shotgun proteomics.
• Group of candidate novel nucleolar
proteins identified by mass spectrometry.
• Isolate corresponding cDNAs.
• Subclone into YFP expression vectors.
• Observe localization in transfected cells
by confocal microscopy.
Fundamental
Molecular Biology
Second Edition
Lisabeth A. Allison
Chapter 16
Genome Analysis:
DNA Typing, Genomics, and Beyond
Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Cover photo: Julie Newdoll/www.brushwithscience.com “Dawn of the
Double Helix”, oil and mixed media on canvas, © 2003
Learning Objectives
• Understand definition, importance and
application of
– GWAS
– HapMap and SNPs
• Personalized Genomics
16.6 Genome-wide association
studies
• All human individuals share genome sequences
that are approximately 99.9% the same.
• The remaining variable 0.1% is responsible for
the genetic diversity between individuals.
• Most common human traits and diseases have a
polygenic pattern of inheritance.
• This means that DNA sequence variants at
many genetic loci influence the phenotype.
• Genome-wide association studies (GWAS) have
identified more than 3000 variants associated
with 150 human traits.
• Example: Hundreds of genetic variants in at
least 180 loci influence adult height.
• Projects investigating cancer genomes and the
genomes of people with diabetes, Alzheimer’s
disease, Crohn’s disease, and other disorders
are under way.
• This type of meta-analysis screens
databases of single nucleotide
polymorphisms, or copy number variants,
to test for the association of a particular
trait with each polymorphism.
Genome Wide Association
Studies GWAS
Single nucleotide polymorphisms
• Two or more possible nucleotides occur
at a specific mapped location in a
genome.
• e.g. ATGCCTA or ATGCTTA
• Must occur in at least 1% of the
population
• Most are not associated with a disease
• Occur every 100-300 bp
• Map of SNPs can be used to scan the
human genome for haplotypes
associated with common diseases.
– e.g. Late onset Alzheimer’s disease
• Haplotypes are patterns of sequence
variation; i.e., stretches of DNA
containing a distinctive set of alleles.
Mapping disease-associated SNPs:
Alzheimer’s disease
• Two SNPs in the apolipoprotein E gene
result in three possible alleles.
• An individual with at least on apo4 (E4)
allele has a greater chance of developing
Alzheimer’s.
Population Studies Can Identify
Associated Alleles
Genes polymorphisms and
human behavior
Human behavior
•
Personality
•
Temperament
•
Cognitive style
•
Psychiatric disorders
Oversimplified model of human behavior
•
Direct linear relationship between individual
genes and behavior.
More accurate model
•
Complex gene networks and multiple
environmental factors affect brain development
and function, which in turn will influence
behavior.
• Heredity definitely plays some role in
behavior but DNA is not destiny.
• In general, be wary of announcements
in the popular media about scientists
finding “the gene” for an aspect of
human behavior.
Why is there a lack-of-progress in finding
“behavior” genes?
•
Behavioral traits are polygenic.
•
Gene-environment interactions.
•
Behavioral traits tend to be inexactly defined.
•
Sample bias.
•
Inadequate sample size.
Polymorphism and Behavior
Aggressive, impulsive,
and violent behavior
•
Family, twin, and adoption studies have
suggested heritability of 0% to >50% for a
predisposition to violent behavior.
•
The case of the “extra” Y chromosome.
•
Polymorphism in the transcriptional control
region of the monoamine oxidase A (MAOA)
gene.
MAOA functional length polymorphism
• MAOA metabolizes several
neurotransmitters in the brain, such as
dopamine and serotonin.
• Prevents excess neurotransmitters from
interfering with communication among
neurons.
High activity alleles
•
Alleles with 3.5 or 4 copies of the repeat
sequence are transcribed more efficiently and
produce more MAOA enzyme.
Low activity alleles
•
Alleles with 3 or 5 copies of the repeat
sequence are transcribed less efficiently and
produce less MAOA enzyme.
High activity alleles (3.5 or 4 repeats)
• Less likely to develop antisocial behavior.
Low activity alleles (3 or 5 repeats)
• More likely to develop antisocial behavior
if maltreated as children.
All of Us NIH Research
Project
• https://allofus.nih.gov
• All of Us is working to improve health care through
research. Unlike research studies that focus on one
disease or group of people, All of Us is building a
diverse database that can inform thousands of
studies on a variety of health conditions. This creates
more opportunities to:
• Know the risk factors for certain diseases
• Figure out which treatments work best for people of different
backgrounds
• Connect people with the right clinical studies for their needs
• Learn how technologies can help us take steps to be healthier
Personalized Genomics
• https://www.23andme.com/
•
•
•
•
•
Get to know you.
Health and ancestry start here.
Reports on 240+ health conditions and traits
Discover your lineage, find relatives and more
Get updates on your DNA as science advances
• https://en.wikipedia.org/wiki/Comparison_of_DNA_se
quencing_service
• https://www.helix.com/

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