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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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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

What is a “vector?”

What is “cloning” a gene?


Cloning a DNA piece into a plasmid

Plasmid

Open plasmid

Isolate gene

Mix

Ligate plasmid


How?

Use __________________ to cut gene

Use __________________to open plasmid

Use __________________to ligate


Restriction Enzymes (REs)

Enzyme that cuts DNA

Called a “Restriction” enzyme because:


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


Type II REs

RE1

GCATGCCGTACG

RE1

GCCGTA

ATGCCG



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!


Reading primary literature

Primary means of scientific communication

- Build upon existing research

- Do not duplicate existing research

Sharing of ideas for scientific progress


Organization of a scientific paper

Abstract/Summary

Introduction

Results

Discussion

Methods & Materials

References


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


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


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


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!


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!


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.


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?


Example

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

yku REQUIRED for NHEJ

BamHI = 5’ overhang


C. elegans life cycle

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


lineage genes in C. elegans


lin-4 regulates lin-14



Molecular mechanisms

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

lin-4 = NOT protein!

lin-4in intron of another gene!


But what does lin-4 encode?


Molecular mechanisms

lin-4 is an RNA!

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

How does small RNA regulate level of another protein?


Is lin-4 unique?


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?


RNAi

+

+

+

+


Paper 1

RNA interference mechanism = miRNA mechanism

Small RNAs generally regulate genes?


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?


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


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!


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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