QuBi/modules/biol303 - Revision history

imported>Lab: /* Discussion Questions */

2021-03-10T21:39:28Z

Discussion Questions

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	[[File:Parikh-fig.png\|thumb\|frameless\|450px\|'''Figure 6.''' The heat maps represent the patterns of change in standardized mRNA abundance for all the genes in the ''D. discoideum'' and the ''D. purpureum'' genomes. Each row represents an average of 85 genes and each column represents a developmental time point (hours). The colors represent relative mRNA abundances (see scale). The genes are ordered according to their regulation pattern in each species. The black lines divide the transcripts, from top to bottom, into: down-regulated, intermediate regulation and up-regulated. The dendrograms represent the differences between the transcriptomes at each time point. Source: [http://genomebiology.com/2010/11/3/R35 Parikh et al (2010)] ]]		[[File:Parikh-fig.png\|thumb\|frameless\|450px\|'''Figure 6.''' The heat maps represent the patterns of change in standardized mRNA abundance for all the genes in the ''D. discoideum'' and the ''D. purpureum'' genomes. Each row represents an average of 85 genes and each column represents a developmental time point (hours). The colors represent relative mRNA abundances (see scale). The genes are ordered according to their regulation pattern in each species. The black lines divide the transcripts, from top to bottom, into: down-regulated, intermediate regulation and up-regulated. The dendrograms represent the differences between the transcriptomes at each time point. Source: [http://genomebiology.com/2010/11/3/R35 Parikh et al (2010)] ]]
	# What differences and similarities does a RNA-Seq experiment have with a Northern blot?		# What differences and similarities does a RNA-Seq experiment have with a Northern blot?
	# Explain how a time-course experiment like this one could be used for discovering gene networks during development		# Explain how a time-course experiment like this one could be used for discovering gene networks during development.
	# The [http://genomebiology.com/2010/11/3/R35 Parikh et al (2010) paper] concludes that developmental pathway genes are conserved in their expression profiles between the two ''Dictyostelium'' species. Based on the two heat maps ('''Figure 6'''), identify similarities and differences in expression profiles between the two species.		# The [http://genomebiology.com/2010/11/3/R35 Parikh et al (2010) paper] concludes that developmental pathway genes are conserved in their expression profiles between the two ''Dictyostelium'' species. Based on the two heat maps ('''Figure 6'''), identify similarities and differences in expression profiles between the two species.
	# Similar genes (homologs) are found in humans. Do you expect the expression of these genes to be similar during human development? during cancer development?		# Similar genes (homologs) are found in humans. Do you expect the expression of these genes to be similar during human development? during cancer development?

imported>Lab: /* Identify co-regulated genes using correlational distances and cluster analysis */

2021-03-10T21:38:24Z

Identify co-regulated genes using correlational distances and cluster analysis

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	# Understand cluster analysis: [http://media.hhmi.org/biointeractive/click/microarray_analyzing/12.html Watch an HHMI slide presentation] on how to group genes and samples by their overall similarity in gene expression levels		# Understand cluster analysis: [http://media.hhmi.org/biointeractive/click/microarray_analyzing/12.html Watch an HHMI slide presentation] on how to group genes and samples by their overall similarity in gene expression levels
	# In the "Hierarchical Clustering" Panel, choose the "Pearson Correlation" for "Distance Function"		# In the "Hierarchical Clustering" Panel, choose the "Pearson Correlation" for "Distance Function"
	# Choose "Average Linkage" for "Linkage" and your choice of color gradient		# Choose "Average Linkage" for "Linkage" and your choice of color gradient.
	# Screen-capture the heatmap and record your answers to the following questions		# Screen-capture the heatmap and record your answers to the following questions
	## What is represented by each row?		## What is represented by each row?

imported>Lab: /* Explore expression profiles of individual genes using dictyExpress */

2021-03-10T21:37:52Z

Explore expression profiles of individual genes using dictyExpress

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	# In the "Experiment and Gene Selection" panel, select "1. D. discoideum vs D. purpurem, Parikh A et.al., D. discoideum." This will select the experiment you read about. Make sure it is highlighted before you do the next steps.		# In the "Experiment and Gene Selection" panel, select "1. D. discoideum vs D. purpurem, Parikh A et.al., D. discoideum." This will select the experiment you read about. Make sure it is highlighted before you do the next steps.
	# In the "Experiment and Gene Selection" panel, type in a Gene Name from Group A (e.g., acrA) in the area under “Genes.”		# In the "Experiment and Gene Selection" panel, type in a Gene Name from Group A (e.g., acrA) in the area under “Genes.”
	# Click "Update Selection." A plot should generate in the "Expression Time Course" panel. Screen shot this plot in your notebook/Word Document/PowerPoint file (Hints: Check the "Legend" box to show the gene’s name on the plot. Click the lower right arrow to expand the plot to full screen as needed. You can move around the windows as well by dragging and dropping the window near the title of the window. For example, you can drag and drop the ~~“expression~~ Time Courses” window to the center of the screen if you wish).		# Click "Update Selection." A plot should generate in the "Expression Time Course" panel. Screen shot this plot in your notebook/Word Document/PowerPoint file (Hints: Check the "Legend" box to show the gene’s name on the plot. Click the lower right arrow to expand the plot to full screen as needed. You can move around the windows as well by dragging and dropping the window near the title of the window. For example, you can drag and drop the “Expression Time Courses” window to the center of the screen if you wish).
	# Is this gene up- or down-regulated during development?		# Is this gene up- or down-regulated during development?
	# Repeat the above for the other genes (15 in total).		# Repeat the above for the other genes (15 in total).

imported>Lab: /* Explore expression profiles of individual genes using dictyExpress */

2021-03-10T21:36:53Z

Explore expression profiles of individual genes using dictyExpress

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	===Explore expression profiles of individual genes using [http://www.dictyExpress.org dictyExpress]===		===Explore expression profiles of individual genes using [http://www.dictyExpress.org dictyExpress]===
	# Click on the website for dictyExpress and "Run dictyExpress (RNA-seq)"		# Click on the website for dictyExpress and "Run dictyExpress (RNA-seq)."
	# A tutorial may start if this is your first time using the website. Feel free to do the tutorial. If you do not want to do the tutorial, close the tutorial box.		# A tutorial may start if this is your first time using the website. Feel free to do the tutorial. If you do not want to do the tutorial, close the tutorial box.
	# In the "Experiment and Gene Selection" panel, select "1. D. discoideum vs D. purpurem, Parikh A et.al., D. discoideum." This will select the experiment you read about. Make sure it is highlighted before you do the next steps.		# In the "Experiment and Gene Selection" panel, select "1. D. discoideum vs D. purpurem, Parikh A et.al., D. discoideum." This will select the experiment you read about. Make sure it is highlighted before you do the next steps.

imported>Lab: /* Explore expression profiles of individual genes using dictyExpress */

2021-03-10T21:36:36Z

Explore expression profiles of individual genes using dictyExpress

← Older revision		Revision as of 21:36, 10 March 2021
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	===Explore expression profiles of individual genes using [http://www.dictyExpress.org dictyExpress]===		===Explore expression profiles of individual genes using [http://www.dictyExpress.org dictyExpress]===
	# ~~Choose~~ the ~~2nd Box:~~ "Run dictyExpress (RNA-seq)"		# Click on the website for dictyExpress and "Run dictyExpress (RNA-seq)"
	# In the "~~Species~~" panel, select "D. ~~dicscoideum~~"		# A tutorial may start if this is your first time using the website. Feel free to do the tutorial. If you do not want to do the tutorial, close the tutorial box.
	# In the "Gene Selection" panel, type in a Gene Name from Group A (e.g., acrA)~~, (DON'T copy and paste; when~~ the ~~gene is found, highlight it and press enter)~~		# In the "Experiment and Gene Selection" panel, select "1. D. discoideum vs D. purpurem, Parikh A et.al., D. discoideum." This will select the experiment you read about. Make sure it is highlighted before you do the next steps.
	# Click "Update" ~~and save the~~ plot in the "Expression ~~Profile~~" panel in your notebook (~~'''~~Hints~~'''~~: Check the "Legend" box to show ~~gene~~ name. Click the ~~top~~ right ~~symbol~~ to expand to full screen. ~~Use~~ the ~~program "Grab" in Apple~~ to ~~capture a~~ screen.)		# In the "Experiment and Gene Selection" panel, type in a Gene Name from Group A (e.g., acrA) in the area under “Genes.”
	# Is this ~~genes~~ up- or down-regulated during development?		# Click "Update Selection." A plot should generate in the "Expression Time Course" panel. Screen shot this plot in your notebook/Word Document/PowerPoint file (Hints: Check the "Legend" box to show the gene’s name on the plot. Click the lower right arrow to expand the plot to full screen as needed. You can move around the windows as well by dragging and dropping the window near the title of the window. For example, you can drag and drop the “expression Time Courses” window to the center of the screen if you wish).
	# Repeat the above for the other genes		# Is this gene up- or down-regulated during development?
	# Group genes by their similarity in expression profiles. How many such groups you ~~could~~ identify? For each group, describe the pattern and speculate on their functions.		# Repeat the above for the other genes (15 in total).
			# Group genes by their similarity in expression profiles. How many such groups can you identify? For each group, describe the pattern and speculate on their functions.
	----		----

imported>Lab: /* Introduction */

2021-03-10T21:31:05Z

Introduction

← Older revision		Revision as of 21:31, 10 March 2021
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	Traditionally, gene expressions are studied one gene at a time using blotting techniques. For example, in a '''Northern Blot''' experiment ('''Figure 2a'''), the whole messenger RNA (mRNA) content of a cell is extracted and loaded on a solid gel slab. Different mRNA molecules are then separated using electrophoresis and transferred to a nitrocellulose sheet. To identify if a gene is expressed, a radioactively (or fluorescently) labeled oligonucleotide probe that is specific to the gene sequence is applied to the sheet. If the gene is expressed, the probe will hybridize with a specific mRNA molecule and a black band will appear on an Xray film. Other blotting techniques for detecting gene expression include '''Southern Blot''', in which mRNAs in a cell are reverse transcribed to their complementary DNA (cDNA) before being hybridized with gene-specific oligo-nucleotide probes. In a Western Blot experiment, the protein product (instead of the mRNA intermediate) of a gene is probed using antibodies (instead of the oligonucleotide probes).		Traditionally, gene expressions are studied one gene at a time using blotting techniques. For example, in a '''Northern Blot''' experiment ('''Figure 2a'''), the whole messenger RNA (mRNA) content of a cell is extracted and loaded on a solid gel slab. Different mRNA molecules are then separated using electrophoresis and transferred to a nitrocellulose sheet. To identify if a gene is expressed, a radioactively (or fluorescently) labeled oligonucleotide probe that is specific to the gene sequence is applied to the sheet. If the gene is expressed, the probe will hybridize with a specific mRNA molecule and a black band will appear on an Xray film. Other blotting techniques for detecting gene expression include '''Southern Blot''', in which mRNAs in a cell are reverse transcribed to their complementary DNA (cDNA) before being hybridized with gene-specific oligo-nucleotide probes. In a Western Blot experiment, the protein product (instead of the mRNA intermediate) of a gene is probed using antibodies (instead of the oligonucleotide probes).

	After the genomic revolution since the 1990s, it became possible to study the expression of all genes in a cell at once using ''high-throughput'' techniques. Detecting the expression profiles of a whole genome was made possible by the availability of the whole genome sequences of bacteria, yeasts, and humans. The '''DNA microarray''' ('''Figure 2b''') is one such high throughput technique. In contrast to the Northern Blot technique in which the mRNA sample is fixed on a nylon sheet, nucleotide probes for all genes are fixed on a glass slide, creating a “gene chip”. The cellular mRNAs are reverse transcribed into cDNAs labeled with fluorescent dyes, which are then hybridized with the gene chips. After the unattached cDNAs are washed away, the fluorescent intensity remains at each probe location is measured as an indication of the amount of mRNA transcribed from each gene in a genome. The entire cellular RNA content transcribed from a genome is called a '''transcriptome'''. Each DNA microarray reading is therefore essentially a snap shot of the whole genome expression profile of a cell at a particular physiological stage. It is no longer necessary to know or decide beforehand candidate genes to be targets of exploration, as in the traditional blotting techniques.		After the genomic revolution since the 1990s, it became possible to study the expression of all genes in a cell at once using ''high-throughput'' techniques. Detecting the expression profiles of a whole genome was made possible by the availability of the whole genome sequences of bacteria, yeasts, and humans. The '''DNA microarray''' ('''Figure 2b''') is one such high throughput technique. In contrast to the Northern Blot technique in which the mRNA sample is fixed on a nylon sheet, nucleotide probes for all genes are fixed on a glass slide, creating a “gene chip”. The cellular mRNAs are reverse transcribed into cDNAs labeled with fluorescent dyes, which are then hybridized with the gene chips. After the unattached cDNAs are washed away, the fluorescent intensity remains at each probe location is measured as an indication of the amount of mRNA transcribed from each gene in a genome. The entire cellular RNA content transcribed from a genome is called a '''transcriptome'''. Each DNA microarray reading is therefore essentially a snap-shot of the whole genome expression profile of a cell at a particular physiological stage. It is no longer necessary to know or decide beforehand candidate genes to be targets of exploration, as in the traditional blotting techniques.

	Most recently, direct sequencing of the whole mRNA content of a cell using the so-called '''RNA-SEQ technology''' ('''Figure 3''') provides an alternative and even more accurate way of obtaining the transcriptome of a cell. Unlike the microarray technology, the RNA-SEQ technology allows ''de novo'' discovery of transcribed genes since it does not rely on a pre-defined DNA probes. Another major advantage of the RNA technology is its ability to detect splice variants, which are differentially spliced exons of the same gene.		Most recently, direct sequencing of the whole mRNA content of a cell using the so-called '''RNA-SEQ technology''' ('''Figure 3''') provides an alternative and even more accurate way of obtaining the transcriptome of a cell. Unlike the microarray technology, the RNA-SEQ technology allows ''de novo'' discovery of transcribed genes since it does not rely on a pre-defined DNA probes. Another major advantage of the RNA technology is its ability to detect splice variants, which are differentially spliced exons of the same gene.

imported>Lab: /* Introduction */

2021-03-10T21:30:37Z

Introduction

← Older revision		Revision as of 21:30, 10 March 2021
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	Traditionally, gene expressions are studied one gene at a time using blotting techniques. For example, in a '''Northern Blot''' experiment ('''Figure 2a'''), the whole messenger RNA (mRNA) content of a cell is extracted and loaded on a solid gel slab. Different mRNA molecules are then separated using electrophoresis and transferred to a nitrocellulose sheet. To identify if a gene is expressed, a radioactively (or fluorescently) labeled oligonucleotide probe that is specific to the gene sequence is applied to the sheet. If the gene is expressed, the probe will hybridize with a specific mRNA molecule and a black band will appear on an Xray film. Other blotting techniques for detecting gene expression include '''Southern Blot''', in which mRNAs in a cell are reverse transcribed to their complementary DNA (cDNA) before being hybridized with gene-specific oligo-nucleotide probes. In a Western Blot experiment, the protein product (instead of the mRNA intermediate) of a gene is probed using antibodies (instead of the oligonucleotide probes).		Traditionally, gene expressions are studied one gene at a time using blotting techniques. For example, in a '''Northern Blot''' experiment ('''Figure 2a'''), the whole messenger RNA (mRNA) content of a cell is extracted and loaded on a solid gel slab. Different mRNA molecules are then separated using electrophoresis and transferred to a nitrocellulose sheet. To identify if a gene is expressed, a radioactively (or fluorescently) labeled oligonucleotide probe that is specific to the gene sequence is applied to the sheet. If the gene is expressed, the probe will hybridize with a specific mRNA molecule and a black band will appear on an Xray film. Other blotting techniques for detecting gene expression include '''Southern Blot''', in which mRNAs in a cell are reverse transcribed to their complementary DNA (cDNA) before being hybridized with gene-specific oligo-nucleotide probes. In a Western Blot experiment, the protein product (instead of the mRNA intermediate) of a gene is probed using antibodies (instead of the oligonucleotide probes).

	After the genomic revolution since 1990s, it became possible to study the expression of all genes in a cell at once using ''high-throughput'' techniques. Detecting the expression profiles of a whole genome was made possible by the availability of the whole genome sequences of bacteria, yeasts, and humans. The '''DNA microarray''' ('''Figure 2b''') is one such high throughput technique. In contrast to the Northern Blot technique in which the mRNA sample is fixed on a nylon sheet, nucleotide probes for all genes are fixed on a glass slide, creating a “gene chip”. The cellular mRNAs are reverse transcribed into cDNAs labeled with fluorescent dyes, which are then hybridized with the gene chips. After the unattached cDNAs are washed away, the fluorescent intensity remains at each probe location is measured as an indication of the amount of mRNA transcribed from each gene in a genome. The entire cellular RNA content transcribed from a genome is called a '''transcriptome'''. Each DNA microarray reading is therefore essentially a snap shot of the whole genome expression profile of a cell at a particular physiological stage. It is no longer necessary to know or decide beforehand candidate genes to be targets of exploration, as in the traditional blotting techniques.		After the genomic revolution since the 1990s, it became possible to study the expression of all genes in a cell at once using ''high-throughput'' techniques. Detecting the expression profiles of a whole genome was made possible by the availability of the whole genome sequences of bacteria, yeasts, and humans. The '''DNA microarray''' ('''Figure 2b''') is one such high throughput technique. In contrast to the Northern Blot technique in which the mRNA sample is fixed on a nylon sheet, nucleotide probes for all genes are fixed on a glass slide, creating a “gene chip”. The cellular mRNAs are reverse transcribed into cDNAs labeled with fluorescent dyes, which are then hybridized with the gene chips. After the unattached cDNAs are washed away, the fluorescent intensity remains at each probe location is measured as an indication of the amount of mRNA transcribed from each gene in a genome. The entire cellular RNA content transcribed from a genome is called a '''transcriptome'''. Each DNA microarray reading is therefore essentially a snap shot of the whole genome expression profile of a cell at a particular physiological stage. It is no longer necessary to know or decide beforehand candidate genes to be targets of exploration, as in the traditional blotting techniques.

	Most recently, direct sequencing of the whole mRNA content of a cell using the so-called '''RNA-SEQ technology''' ('''Figure 3''') provides an alternative and even more accurate way of obtaining the transcriptome of a cell. Unlike the microarray technology, the RNA-SEQ technology allows ''de novo'' discovery of transcribed genes since it does not rely on a pre-defined DNA probes. Another major advantage of the RNA technology is its ability to detect splice variants, which are differentially spliced exons of the same gene.		Most recently, direct sequencing of the whole mRNA content of a cell using the so-called '''RNA-SEQ technology''' ('''Figure 3''') provides an alternative and even more accurate way of obtaining the transcriptome of a cell. Unlike the microarray technology, the RNA-SEQ technology allows ''de novo'' discovery of transcribed genes since it does not rely on a pre-defined DNA probes. Another major advantage of the RNA technology is its ability to detect splice variants, which are differentially spliced exons of the same gene.

imported>Lab: /* Objectives */

2021-03-10T21:29:56Z

Objectives

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	==Objectives==		==Objectives==
	# Understand the RNA-SEQ technology and its use in ~~genomewide~~ identification of gene functions.		# Understand the RNA-SEQ technology and its use in genome-wide identification of gene functions.
	# Be able to identify co-expressed and co-repressed genes based on time-course gene expression data.		# Be able to identify co-expressed and co-repressed genes based on time-course gene expression data.
	----		----

imported>Lab at 19:21, 26 February 2021

2021-02-26T19:21:37Z

← Older revision		Revision as of 19:21, 26 February 2021
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	==References & Resources==		==References & Resources==
	# [http://dictybase.org/ DictyBase: a database of Dictyostelium genes]		#[https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-017-1706-9 DictyExpress: a web-based platform for sequence data management and analytics in Dictyostelium and beyond]
			#[http://dictybase.org/ DictyBase: a database of Dictyostelium genes]
	# [http://www.dictyExpress.org DictyExpress: a web application to analyze gene expressions in Dictyostelium species]: [https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-017-1706-9 Paper]		# [http://www.dictyExpress.org DictyExpress: a web application to analyze gene expressions in Dictyostelium species]: [https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-017-1706-9 Paper]
	# [http://genomebiology.com/2010/11/3/R35 Parikh et al (2010). Conserved developmental transcriptomes in evolutionarily divergent species. ''Genome Biology'', 11:R35]		# [http://genomebiology.com/2010/11/3/R35 Parikh et al (2010). Conserved developmental transcriptomes in evolutionarily divergent species. ''Genome Biology'', 11:R35]
	# [http://www.ncbi.nlm.nih.gov/pubmed/19015660 Wang, Gerstein, and Snyder (2009). RNA-Seq: a revolutionary tool for transcriptomics. Natural Review of Genetics]		# [http://www.ncbi.nlm.nih.gov/pubmed/19015660 Wang, Gerstein, and Snyder (2009). RNA-Seq: a revolutionary tool for transcriptomics. Natural Review of Genetics]
	# [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17637 Description of the experiment and data from the NCBI GEO database]		# [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17637 Description of the experiment and data from the NCBI GEO database]

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imported>Lab: /* Lab Report Grading Policy */

2021-02-26T19:01:02Z

Lab Report Grading Policy

← Older revision		Revision as of 19:01, 26 February 2021
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	==Lab Report Grading Policy==		==Lab Report Grading Policy==
	; '''Introduction''' ''(3 pts)'' : Define transcriptome. List key steps in RNA-SEQ technology. Describe advantages of high-throughput technologies in comparison with traditional gene-by-gene approaches of studying gene function. Your statements are not to be copied from the Lab Manual.		; '''Introduction''' ''(1 pts)'' : Define transcriptome. List key steps in RNA-SEQ technology. Describe advantages of high-throughput technologies in comparison with traditional gene-by-gene approaches of studying gene function. Your statements are not to be copied from the Lab Manual.

	; '''Materials and Methods''' ''(4 pts)'': Describe experimental procedures of the study that have produced these gene expression data by reading [http://genomebiology.com/2010/11/3/R35 this paper] and [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17637 this experimental report]. Answer the following questions:		; '''Materials and Methods''' ''(1 pts)'': Describe experimental procedures of the study that have produced these gene expression data by reading [http://genomebiology.com/2010/11/3/R35 this paper] and [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17637 this experimental report]. Answer the following questions:
	# Name of the two species used in experiments		# Name of the two species used in experiments
	# How many genes were measured for their expression levels?		# How many genes were measured for their expression levels?
	# How many time points, developmental stages, and cell types have been tested?		# How many time points, developmental stages, and cell types have been tested?

	; '''Results''' ''(12 pts)'':		; '''Results''' ''(5 pts)'':
	# Table 2 (annotation for 15 genes)		# Table 2 (annotation for 15 genes)
	# Expression profiles (screen capture for 15 genes)		# Expression profiles (screen capture for 15 genes)
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	# Heat map of 15 genes (screen capture)		# Heat map of 15 genes (screen capture)

	; '''Discussion''' ''(9 pts)'' : Answer the four discussion questions.		; '''Discussion''' ''(2 pts)'' : Answer the four discussion questions.

	; '''Summary/Conclusion''' ''(1 pt)'' : A sentence or two will suffice.		; '''Summary/Conclusion''' ''(1 pt)'' : A sentence or two will suffice.