Chapter 12.1-12.4

signtruculentBiotechnology

Oct 2, 2013 (3 years and 8 months ago)

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Chapter 12

DNA Technology and the Human
Genome

Do Now: 10 minutes

Study Technique


Skim chapter opening pg 230
-
231


1) Define bold words and look at pictures


2) Make a quick outline of main points with at
least 3 spaces in between in one color


3) Use a different color to fill in under each
section with details from the book


4) In a different color come up with two
questions, one easy and one difficult

Should look like this….


Recombinant DNA technology: combining genes from different sources


DNA technology: methods for studying and manipulating DNA


Genomics: Study of genomes based on sequence


Bioinformatics: using math to analyze bio data


Proteomics: study of proteins



Bacteria studies


Joined by pilli (male) passing DNA to “female”


Respond to environment


Lederburg and
T
atum: experiments that mixed genes in E. coli (Recombinant
DNA technology


DNA technology


Introns discovered by this


Lead in to HGP


Human Genome project (more than just humans)


Mapping genomes lead to genomics, bioinformatics, and proteomics


Scientists engineer bacteria for practical purposes (make pesticides, cancer
drugs, AIDS vaccine)


Ethical issues on health



What might Ms. Bell Ask…


Easy: How do bacteria have sex?


Hard: Why is this advantageous for bacteria?



Easy: Describe the difference between
genomics, proteomics, and bioinformatics


Hard: Why are people concerned with
recombinant DNA technology? Explain a
hypothetical situation.


12.1 In nature, bacteria can transfer
DNA in three ways


Gene transfer


DNA from one to another bacteria


Non
-
reproductive


new combination


DNA= closed loop w/protein


Three mechanisms


1) Transformation: uptake from surrounding


Griffith experiment: harmless bacteria take up piece from deadly and became
deadly….


2) Transduction: phage


3) Conjugation: Male donor, pili


Female recipient


Cytoplasmic bridge



New DNA may integrate into recipient






Question… Which type of gene transfer allows
one copy of DNA to peel off and transfer to
recipient?

12.2 Bacteria plasmids can serve as
carriers for gene transfer


F factor


Genes for conjugation


Also contains origin of replication


Acts as the leading end of transferred chromosome


Plasmid


Small circular DNA that contains origin of replication


Some contain F factor


Vector s: Can carry genes other than F factor


R plasmid


Contain enzymes that destroy antibiotics like penicillin or
tetracycline


Act as useful vectors in genetic engineering

LE 12
-
02

Restriction enzyme

recognition sequence

Restriction enzyme

cuts the DNA into

fragments

Addition of a DNA

fragment from

another source

DNA

G


C


A


A


T


T


C


G


G


C


C


G


G


Sticky end

Two (or more)

fragments stick

together by

base
-
pairing

DNA ligase

pastes the strand

Recombinant DNA molecule

G


G


G


G


C


C


C


C


A


A


A


A


A


A


T


T


T


T


T


T


T


T


A


A


C


T


T


A


A


G


How restriction
enzymes work…

LE 12
-
01
-
3

Bacterial

chromosome

Plasmid

Bacterium

Plasmid

isolated

Recombinant DNA

(plasmid)

Recombinant

bacterium

Copies of gene

Clone of cells

Gene for pest

resistance

inserted into

plants

Gene used to alter bacteria

for cleaning up toxic waste

Protein used to dissolve blood

clots in heart attack therapy

Copies of protein

Cell multiplies with

gene of interest

Protein used to

make snow

form at higher

temperature

Plasmid put into

bacterial cell

DNA

Gene of

interest

Gene inserted

into plasmid

DNA

isolated

Cell containing gene

of interest

12.3 Plasmids are used to customize
bacteria: An overview


1) plasmid isolated


2) DNA of interest is obtained


3) DNA inserted


4) Bacteria uptakes plasmid


5) genetically engineered, recombinant bacteria
cloned



Challenge: How can R plasmids be helpful in
terms of recombinant DNA technology?

LE 12
-
03

Isolate DNA

from two sources

Cut both DNAs

with the same

restriction enzyme

Mix the DNAs;

they join by

base
-
pairing

Add DNA ligase

to bond the DNA covalently

Recombinant DNA

plasmid

E. coli

Plasmid

Human cell

Sticky ends

Gene
V

DNA

Gene
V

Put plasmid into bacterium

by transformation

Recombinant

bacterium

Clone the bacterium

Bacterial clone carrying many

copies of the human gene


LE 12
-
04

Recombinant

plasmid

Bacterial

clone

Plasmid library

Phage

clone

Phage library

Recombinant

phage DNA

Genome cut up with

restriction enzyme

or

12.7 Reverse Transcriptase helps make
genes for cloning


Do Now: Turn to page 236 section 12.7


1) Read the section


2) Redraw the picture


3) Describe cDNA and the process of making a
cDNA


4) Why might cDNA of insulin might work
better in E.coli than trying to use a normal
gene?


LE 12
-
05

Cell nucleus

mRNA

Test tube

DNA of

eukaryotic

gene

Reverse transcriptase

cDNA strand

cDNA of gene

(no introns)

RNA

transcript

Exon

Exon

Exon

Intron

Intron

Transcription

RNA splicing

(removes introns)

Isolation of mRNA

from cell and addition

of reverse transcriptase;

synthesis of DNA strand

Breakdown of RNA

Synthesis of second

DNA strand

12.8 Nucleic acid probes identify
clones carrying specific genes


It is difficult to tell if clone contains
________________________


Methods depends on base pairing


Part of the DNA is known


Gene




_________


Probe (radioactive)


_________


Steps:


1) _________________________________


2) Treat (heat or alkali) to ___________________


3) Add radioactive probe (reality ___________________)


4)Autoradiography (develop film to determine radioactivity)


5) Compare to master plate (____________________)

LE 12
-
08

Radioactive

probe (DNA)

Single
-
stranded

DNA

Mix with single
-

stranded DNA from

various bacterial

(or phage) clones

Base pairing

indicates the

gene of interest

12.9 DNA microarrays test for
expression of many genes


DNA microarrays (chips)


Used for______________________________


Measures gene expression at _______________


Steps


______________________


mRNA used to make radioactive ________


Exposed to a chip


Benefits


Thousands of genes tested at once


Each radioactive spot represents gene expression


LE 12
-
09

mRNA

isolated

cDNA made

from mRNA

cDNA applied

to wells

Unbound

cDNA rinsed

away

Each well contains DNA

from a particular gene

Fluorescent

spot

DNA microarray

Nonfluorescent

spot

cDNA

DNA of an

expressed gene


DNA of an

unexpressed gene


Reverse transcriptase

and fluorescent DNA

nucelotides

Actual size

(6,400 genes)

12.10
-

12.11 Gel electrophoresis:


Mixture of DNA

molecules of

different sizes

Power

source

Gel

Completed gel

Longer

molecules

Shorter

molecules

12.12 The PCR method is used to
amplify DNA sequences


Polymerase chain reaction


Steps:


_____________________


DNA is incubated
with____________________
________________________


Benefits:


Each cycle doubles DNA
amount


Takes hours


Can copy specific segment


Starting material does not
need to be purified


PCR: The good and the bad


Even though it is faster, it cannot replace gene
cloning in cells due to occasional errors that
impose limits


Uses:


________________________


________________________

12.13 Most of the human genome
does not consist of genes


Most of our DNA does not code for protein


35,000 genes that code for protein, tRNA and
rRNA


97% is non
-
coding


Types of junk


__________and DNA between genes


Repetitive DNA


Short repeats


Associated with ________________and ends of chromosomes
(chromosome structure)


Telomeres: at the end of chromosomes and related to cell death and
cancer


Cells that can regenerate telomeres can “_______________”


Might deal with gene ___________________


Some nervous diseases caused by abnormal stretches of triplet


Huntington's: _____ repeat in coding region


Long Repeats: function unknown


Example: Transposons: genes that can jump from one location to the next
(researcher Barbara McClintock)

»
Cut and paste or ______, cut and paste (leaves sequence behind)

»
Natural mutagen to generate ________________

»
Implicated in some cancer



LE 12
-
11b

Longer

fragments

1

x

Shorter

fragments

w

y

y

z

2

LE 12
-
12a

Defendant’s

blood

Blood from

defendant’s clothes

Victim’s

blood

LE 12
-
13

Cloned gene

(normal allele
)

Insert normal gene

into virus

Viral nucleic

acid

Retrovirus

Infect bone marrow

cell with virus

Viral DNA inserts

into chromosome

Bone marrow

cell from patient

Bone

marrow


Inject cells

into patient

LE 12
-
14

Initial

DNA

segment

Number of DNA molecules

1


2


4


8


12.4
-
12.6 Recombinant Lab


Add picture from each….