powerpoint genetic engineering

roachavocadoBiotechnology

Dec 14, 2012 (4 years and 11 months ago)

241 views

Genomics & Biotechnology,
Chapter 20 Campbell’s Bio ed 7

VIEW IN SLIDE SHOW MODE

What is Genomics?

SEQUENCING OF GENOMES, FOLLOWED BY
APPLICATION OF THE DATA EITHER TO:




FURTHER RESEARCH OF THE GENOME & ITS EXPRESSION

Or


TO ACHIEVE HUMAN GOALS


1

HOW TO USE THIS LESSON

Work through the slides, exiting to run animations at the
Campbell’s 7
th

edition website. A couple of other sites will
also be useful, especially
www.HHMI.org/biointeractive


A study guide is attached to this week on the course
webpage, also. After each slide, question numbers you can
answer are indicated. Stop and answer these questions
(type into the form and save to your directory) before
moving on to the next slide or activity.

http://wps.aw.com/wps/media/access/Pearson_Default/1663/1703422/login.html

Username


Mayfieldstudent1

password

scienceaccount2009If you can’t access the login page directly,
use this strategy:

http://www.campbellbiology.com/

Click on the picture of your textbook

Now log in as above

2

What is “
cloning a gene
”? Why do
scientists clone genes? Fig20
-
2 P 385

Cloning a gene

is the process of
placing a gene sequence in a
piece of DNA or RNA called a
vector
, then
transforming

the
vector into
host cells
:
bacteria, viruses, or yeasts.

Rapid reproduction of either of
these hosts result in rapid
copying
(amplifying)
the
vector with its cloned gene
with every round of host cell
division.*

Run the activity for 20.1, cloning a
gene in bacteria

Complete Study guide #3, #4


3


Run 20.1 activity
applications of DNA technology

examples of how DNA technology is applied



study guide #1 (click on glossary for 2
nd
, 3
rd

terms)


Run 20.5 activity
making decisions about DNA
technology: golden rice



study guide #34


4

Before you can clone a gene, you
must remove it from the
chromosome and place it into a
vector that can transfer it into a
host cell.

Restriction enzymes
allow you to cut sequences
out of DNA or to prepare vectors for new DNA.*


Run activity for 20.1, Restriction enzymes


5

Tools of the trade: Restriction enzymes

Fig 20
-
3 Restriction enzymes cut DNA at specific recognition
sequences and generate “sticky” (complementary single
stranded ends) fragments. These are needed to insert genes
into vectors.*
Do study guide #5


6

Note restriction enzymes recognize and cut sequences
having the same sequence in the 5’

3’ direction on
both complementary strands.


The ends are sticky
because they
complement the ends
of any other piece of
DNA cut with the same
restriction enzyme.


The complementary
base pairs align and H
bond.


Some restriction
enzymes will not cut
DNA that has been
modified by
methylation (so
heterochromatin is hard
to cut).

7

Once cut out of the original
chromosome, the gene must be
prepared for transfer into the new
host cell.


It must be ligated into a
piece of DNA,
a vector,
that can transfer it into
the new host.


If the vector and gene
are cut with the same
restriction enzyme, the
enzyme ligase
will join
them together.*


8

Tools of the trade: What is a
vector
?

A vector is a piece of DNA that can shuttle DNA from one
organism to another.

Vectors contain sequences that allow for
selection

(differential survival) of cells containing them along with
inserted genes.

These are the vectors you must be able to describe:


Bacterial plasmids (like the Ti plasmid)

Retroviral vectors

Attenuated retroviral vectors

Yeast artificial chromosomes*

9

Tools of the trade: What is a
vector
?

A vector is a piece of DNA that can shuttle DNA
from one organism to another.


Plasmid

small circular DNA element found
in the cytoplasm of bacteria, some
protists, and sometimes plants. In
bacteria, these replicate when the host
chromosome replicates.

Ti plasmid

plasmid normally found in a
bacterium causing plant tumors (galls).

Ti plasmids
allow high efficiency
transformation of plants. Plants can be
propogated asexually (by rooting
cuttings), facilitating gene transfer in plant
populations*
do study guide #33

10

Tools of the trade: Bacterial plasmid vectors

Bacterial plasmids commonly carry antibiotic resistance genes that
allow only cells that contain the plasmid to survive selection.

Many different genetically modified plasmids are
available for purchase.

These can be purchased to code particular
antibiotic resistance (for selection)and to
allow cloning of genes with different terminal
restriction sequences at the multi
-
cloning site.

Weakness: Plasmids an’t carry whole genes with
regulatory regions & introns (genes too large);
bacterial promoters don’t bind normal
eukaryotic transcription factors, so expression
may not be regulated normally if used in gene
therapy.
Also, bacterial hosts don’t carry out
alternative splicing
.*

Do study guide #2, #6

11

Parts of a
plasmid



Rerun 20.1 activity cloning a gene in bacteria


Antibiotic resistance gene
(codes an enzyme
that destroys an antibiotic normally lethal to
bacteria)

allows only cells transformed
with the plasmid with this resistance gene to
survive in the presence of that antibiotic.


Reporter gene
(allows cells having plasmids
with inserted genes to be distinguished from
cells whose plasmids are not carrying a
cloned gene) e.g., beta galactosidase LacZ

keep the host that are
not blue
in presence
of Xgal substrate


Multi
-
cloning site
inside the reporter gene

restriction enzyme cutting sequences allow
the gene of interest to be inserted

if a gene
is inserted, the reporter gene is inactivated.*

12

How do you
transform

bacteria?


Make bacteria competent for plasmid uptake
.
That is, grow them to log phase

that is, to the
point at which the population is growing
exponentially and no limiting factors like
competition for food or space are present.

Then, Use CaCl
2

to burst small holes in the
bacterial cell walls

and add plasmids.

Heat over optimal temperatures for 90 seconds to
activate heat shock proteins (SOS) proteins to
repair the holes.


13

What is a colony? Clones of the same single
bacterium that survived selection in the antibiotic.
Pick the transformed, insert carrying colonies, then
grow large numbers of these. Test them for accuracy
of insertion (correct direction) and accuracy of
sequence (correct size and nucleotide sequence)



14

Run 20.1 investigation: How can antibiotic resistant
plasmids transform E coli?


At

www.
hhmi
.org/bio
interactive

,
go to animations,
DNA, genetic engineering, and play the short
animation on genetic engineering.


Answer study guide #7



15

Phages may also be used as vectors
to transfer DNA into bacteria.



Phages are viruses specific to bacteria.



These are usually used in constructing
DNA
libraries

(covered later in the chapter).


16

Tools of the trade: vectors

retroviruses as vectors for eukaryotic cells



Retroviruses infect hosts cells whose receptors match
binding sites on their capsids.

Retroviruses often contain very strong
promoters (lots of expression of the
gene) that are able to bind eukaryotic
transcription factors AND they infect cells
with high efficiency. This makes them
good vectors for
gene therapy
.

Disadvantages:

Inserted genes can’t be more than a few
thousand base pairs long.

If a retrovirus is used in gene therapy, then it
must be modified to prevent infection of
unintended cells in organisms. These are
called
attenuated retroviral vectors.


17

Tools of the trade:

Yeast Artificial Chromosomes YAKs

Yeast artificial chromosomes(YAKs) are often the best choice for
cloning of eukaryotic genes and for transferring them in gene
therapy because:

1 YAKS can transfer much longer sequences than plasmids and
retroviruses (these max out at a few thousand base pairs, but YAKs
can transfer 10’s of thousands of nucleotides). Eukaryotic genes have
long upstream and downstream regulatory regions and introns, so
these genes can be up to millions of base pairs long!


2 YAKS have eukaryotic promoters, so often these are necessary to bind
the transcription factors present in a mammalian cell. If the vector is
to be used in gene therapy, then this eukaryotic gene expression is
necessary.


18

Review: How is a gene cloned into a plasmid vector then amplified in
host bacterial cells?

Run:
http://highered.mcgraw
-
hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.html

Run
http://highered.mcgraw
-
hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_2.html


19

Now that you’ve obtained colonies that carry
plasmids with inserted genes, how do you confirm
that the gene is the correct gene, is oriented
correctly, and is not damaged in sequence?

Use Restriction fragment analysis OR PCR
analysis to confirm size and orientation.


Use either DNA sequencing or high stringency
probe hybridization (only very accurate
complementary base pairing probes adhere at
high temperature and salt concentration)

20

Restriction fragment analysis allows determination of
whether a host carries plasmids having the size and
orientation and overall structure needed.

Plasmids isolated from selected colonies are cut with
restriction enzymes matching sites in either the plasmid
OR the insert OR both.

Because the sequence of both are known (restriction
maps), it is possible to predict the sizes of DNA
fragments expected.

Gel electrophoresis is used to separate the fragments and
determine their size.


we’ll do simple problems like


this in class

I couldn’t find simple


tutorials on line.

21

Gel electrophoresis
allows you to find the size of a piece of DNA by
comparing it to the sizes of known pieces of DNA (called
DNA size
standards
). DNA
--
negative charge
--
runs from negative pole to positive
pose of gel. Longer pieces travel slower than shorter pieces. P 393
Run 20.2 activity Gel electrophoresis of DNA.

Do study guide questions #15, 16

22

Often mutations in genes interupt restriction
enzyme recognition sites, creating
restriction
fragment polymorphisms (RFLP
s). By cutting DNA
with restriction fragments and comparing size to
known expected sizes, a person’s DNA can be
tested to see whether he carries a disease causing
allele. P 393. For example, this disease allele
would produce smaller pieces of DNA than the
typical allele.

23

Figure 20.9 p 393 shows how to use RFLP analysis to test
for presence of sickle cell allele

When a fragment of DNA carrying the
hemoglobin gene is cut with the
restriction enzyme DdeI, it
produces 2 short pieces (175 bp
and 201 bp and 1 large piece of
DNA), but the sickled allele
produces a larger 376 bp fragment
because it is missing one DdeI site.

Run Activity: Analyzing DNA
Fragments Using Gel
Electrophoresis


Run

Investigation: How Can Gel
Electrophoresis Be Used to Analyze
DNA
?

Do study guide question #17, 18


24

Analyze your ability to interpret
restriction maps


Go to Lab Bench at the Campbell’s website.

Work part II of #6 molecular biology & answer
the questions that go with it. You should write
these into your study guide, rather than typing
them into the online lab report.

25

Another way to analyze genes by the sizes of DNA fragments
they produce is
Polymerase chain reaction (PCR)

P 391 shows a diagram of PCR

Go to
www.hhmi.org/biointeractive
, then go to animations,
DNA, and run Polymerase chain reaction.


26

By choosing PCR primers
complementing correct
sequence for where your
inserted gene is ligated to the
vector, DNA fragment size of the
PCR shows whether a gene
inserted to the plasmid is the
right size and orientation.

PCR fragments that complement
ends of a gene segment carrying
a disease causing mutation
generate products whose size
show whether or not a person
carries a disease causing allele.

Run:
http://highered.mcgraw
-
hill.com/sites/0072556781/student_vi
ew0/chapter14/animation_quiz_6.ht
ml

Do study guide questions 12, 13


27

The most important feature of PCR is that it very rapidly
and cheaply copies DNA, allowing one or two copies to
be amplified to billions within a few hours.


PCR can be used for DNA sequencing.

PCR can be used to replace RFLP analysis.

PCR can be used to add needed restriction
fragments to genes so that you can use any
vector’s multicloning site.


Kerry Mullis
received the Nobel prize for his
invention of PCR technique.


28

How do you find a DNA fragment that
contains the gene you need?

Southern blots
transfer DNA from Gels to specially
positively charged paper (nitrocellulose). Page 395

Labeled Probes
can be used to find the fragment that
contains the gene of interest.

Run: http://highered.mcgraw
-
hill.com/sites/0072556781/student_view0/chapter14/ani
mation_quiz_4.html


Probes used to find a particular gene are single stranded
pieces of DNA or RNA having a sequence unique to the
gene you need. The label will be seen only above the
fragment that complements the probe.

29

Labeled Probe

single stranded piece of
DNA OR RNA that is labeled (e.g.,
radioactive or colored)

here shown in
yellow

will only complement the gene
of interest. P 388

30

Analyze your knowledge of blotting
applications and probes

Run

http://highered.mcgraw
-
hill.com/sites/0072556781/student_view0/ch
apter14/animation_quiz_5.html


Answer study guide questions 9, 10, 19, 20

31

Making & probing a Southern blot p395

32

Blots are used in forensic analysis for identifying
parents, lost persons, suspects, etc.

Run activity 20.5 DNA fingerprinting

In this case, probes recognizes repetitive
elements of DNA whose location in the
genome varies in the genome, such that the
length of fragments hybridizing to each probe
is highly variable in the population.

Tested with enough restriction enzymes, the size
pattern of DNA bands hybridizing to these
probes is unique for each person (probably).
Fig 20.7 p 405

33

The Ultimate test for whether you’ve correctly
cloned a gene is to
sequence the DNA
insert

Run: http://www.dnalc.org/resources/animations/sangerseq.html
http://www.hhmi.org/biointeractive/dna/DNAi_human_genome_seq.html

Run:
http://www.hhmi.org/biointeractive/dna/DNAi_sanger_sequencing.html

Run:
http://www.hhmi.org/biointeractive/dna/DNAi_shotgun_seq.html

Answer study guide 21, part 1


Genomic Libraries

a

single tube of viral supernatant (like
lambda
λ

phage DNA) or of bacteria, like E. coli, contains
vectors whose inserts include every sequence in the genome,
cut into fragments. Use it to obtain a gene, ready to use.

1 Isolate genomic DNA (total DNA) from an organism’s cells
(for example, from crushed muscle)

2 Cut the DNA with a restriction enzyme, generating many
fragments with the same sticky ends


e.g., when a “6 cutter enzyme”
--
like EcoR1
--
whose
restriction site contains 6 nucleotides is used, the human
genome is cut into pieces 20K base pairs long.

3 Cut the vector (usually
λ

phage with the same R enzyme),
then ligate in the entire collection of inserts.

4 infect bacterial hosts (transduction), select, and freeze the
entire culture until time to screen it for a certain gene.

35

Genomics involves sequencing entire genomes,
then applying the info

use genomic libraries to
get the DNA fragments used in sequencing
.

cDNA Libraries

a

single tube of viral supernatant (like
lambda
λ

phage) or E. coli contains vectors whose inserts
include DNA complementary to RNA being expressed in
the cell at the time RNA was collected. Use it to compare
gene expression in the same organism, but in different
differentiated cell types, in the same cell type under
different conditions, etc.

1
Obtain total RNA Or mRNA from cells

2
Add
retroviral reverse transcriptase
to reverse
transcribe the RNA molecules into complementary
DNA (cDNA)

no introns, shorter than genomic
DNA inserts + whole transcript on a single fragment

3
Cut cDNA’s with same Restriction enzyme as the
λ
DNA, then ligate, transduce into bacteria, keep the
culture until ready to screen for particular genes.

37

Screening libraries for genes

1 Use library viral supernatant (liquid collected and frozen) to infect E
coli.

2 Spread cells onto agar plates to create a uniform
“lawn of bacteria”.

3 Cells containing phage will multiply & be surrounded on the plate by
their clones. As a
lytic

viral life cycle is completed, the phage will
kill bacteria, leaving
plaques

(clear spots where cytoplasm of dead
cells is filled with phage).

4 Use filter paper to collect a replica of all the plaques on the plate

screen the paper for which gene or
cDNA

is cloned into the virus of
each plaque.

5
Heat the replicas to make DNA single stranded.

6
Treat paper with heated single stranded
labeled probes
.

7
Collect plaques at matching sites to
hybridized probe
, then expand
to amplify the copies of the phage to use it in research.

38

DNA microarrays
allow rapid screening
for genes important in phenotype.




RUN:
http://www.hhmi.org/biointeractive/genomics/s
m_microarrays.html

Watch
http://www.hhmi.org/biointeractive/genomics/
gene_chips.html







Do study guide question 22 and the 2
nd

half of
21

40