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Last class. Separating and visualizing DNA. Experimental design. Working with DNA. Genes/regions of interest in larger pieces of DNA. Isolate gene/region of interest to study it/products. Examples of things you can do with DNA: - - -. First step:. Need DNA “scissors”!. Cloning.

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Separating and visualizing dna

Last class

Separating and visualizing DNA

Experimental design


Separating and visualizing dna

Working with DNA

Genes/regions of interest in larger pieces of DNA

Isolate gene/region of interest to study it/products

Examples of things you can do with DNA:

-

-

-

First step:

Need DNA “scissors”!


Separating and visualizing dna

Cloning

What is a “vector?”

What is “cloning” a gene?


Separating and visualizing dna

Cloning a DNA piece into a plasmid

Plasmid

Open plasmid

Isolate gene

Mix

Ligate plasmid


Separating and visualizing dna

How?

Use __________________ to cut gene

Use __________________to open plasmid

Use __________________to ligate


Separating and visualizing dna

Restriction Enzymes (REs)

Enzyme that cuts DNA

Called a “Restriction” enzyme because:


Separating and visualizing dna

Type II REs

Type II REs “cut” inside dsDNA

Type II REs cut at specific recognition sites

RE site = specific DNA sequence

Almost always a palindromic sequence because:

No length limits for RE site. Usually 4-8 nucleotides


Separating and visualizing dna

Type II REs

RE1

GCATGCCGTACG

RE1

GCCGTA

ATGCCG


Separating and visualizing dna

What can I clarify?


Separating and visualizing dna

Reading primary literature

Reviews, news, etc. ALWAYS biased!

- News: Stem cells will allow us to live forever

- Religious right: Billions of dollars wasted on useless, immoral research

- Research: Stem cells MIGHT be therapeutic

You might want to look at the actual research!

Importance of forming your OWN opinions

- Elections, democracy and political spin!

- Informed decisions!


Separating and visualizing dna

Reading primary literature

Primary means of scientific communication

- Build upon existing research

- Do not duplicate existing research

Sharing of ideas for scientific progress


Separating and visualizing dna

Organization of a scientific paper

Abstract/Summary

Introduction

Results

Discussion

Methods & Materials

References


Separating and visualizing dna

The parts of a paper: Establish rationale

Abstract/Summary

- Brief version of paper

- Main question/s addressed

- Experimental approach

- Main result/s & their significance

Introduction

- Background and context for the research

- Purpose/Hypothesis


Separating and visualizing dna

The parts of a paper: Present data

Results

- Data generated by experiments (Graphs, charts, tables, etc.)

- Explanations to understand results

Methods & Materials

- Science should be reproducible!

- Allows reader to duplicate results


Separating and visualizing dna

The parts of a paper: Put in context, future

Discussion

- Brief summary of results

- Significance

- Future directions

References

- Credit for past work

- Resource for additional information/reading


Separating and visualizing dna

How to read primary literature

Not easy!

- Usually quite jargony

- Practice, practice, practice

Sequential

- Each experiment will build on previous one

Active & Critical

- Research, not text book!

- Conclusions may not be correct!


Separating and visualizing dna

Textbook VS Primary literature

Textbook

Literature

Assumption

Ideas are correct

Ideas are wrong!

How /Method

Unimportant

Critical!

Prior knowledge

Low requirement

High

requirement!

Reading

Passive

Active!


Separating and visualizing dna

Building a knowledge base

“By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme.”

eLife 2013;2:e00278

Glucose–TOR signalling reprograms the transcriptome and activates meristems

Nature 496, 181–186

Read, read, read, read, read.


Separating and visualizing dna

How to read a paper

Main question/s or hypothesis/es of paper

Individual figures

- Purpose of the experiment

- How does data relate to main question/s?

Active & Critical

- Does the data support conclusions?

- Additional experiments?

- Future directions

- Controversy? Paradoxes?


Separating and visualizing dna

Example

Main hypothesis: Yeast SUB1 (PC4) is required for NHEJ

yku REQUIRED for NHEJ

BamHI = 5’ overhang


Separating and visualizing dna

C. elegans life cycle

C. elegans Life Stages from George L. Sutphin and Matt Kaeberlein at Univ. of Washington, Seattle.


Separating and visualizing dna

lineage genes in C. elegans


Separating and visualizing dna

lin-4 regulates lin-14


Separating and visualizing dna

The lineage cascade in C. elegans


Separating and visualizing dna

Molecular mechanisms

lin-14, lin-28, lin-29 were all proteins

lin-4 = NOT protein!

lin-4in intron of another gene!


Separating and visualizing dna

But what does lin-4 encode?


Separating and visualizing dna

Molecular mechanisms

lin-4 is an RNA!

lin-4is a small (~22nt) RNA!

How does small RNA regulate level of another protein?


Separating and visualizing dna

Is lin-4 unique?


Separating and visualizing dna

Paper 1

lin-4 & let-7 Only in C. elegans?

Found let-7-like sequences in other organisms

Small RNAs  Universal regulators?

Small RNAs  How widespread? Roles?


Separating and visualizing dna

RNAi

+

+

+

+


Separating and visualizing dna

Paper 1

RNA interference mechanism = miRNA mechanism

Small RNAs generally regulate genes?


Separating and visualizing dna

Paper 1

Found let-7-like sequences in other organisms

Small RNAs  Universal regulators?

Small RNAs  How widespread? Roles?

How might you identify small RNAs?


Separating and visualizing dna

Your assignment…

Download assignment

Answer each question about indicated figure

Do not go over space limit/Do not change font!

Save/Convert to PDF

Upload to Turnitin

Deadline: 18-Oct-2013 4:00pm


Separating and visualizing dna

Paper reading ~ Lab report reading!

Peer review is nota waste of time!

Read lab reports critically ~ Reading paper!

Gain: process, not just getting feedback

Peer review = Universal career requirement!


Separating and visualizing dna

Lab report 1 and Peer review

Lab report one deadline varies

Due before your lab section

Peer review deadline is 01/28, 11:59pm

Reviews released on 01/29, 12:00am

Cannot do reviews after deadline!!!


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