Biotechnology and Genomics_________________ is the production of identical copies of DNA, cells or organisms

If you think about it, members of a bacterial colony in a sample are clones because they all came from division of the same cell

____________ twins are even clonesSingle embryo separate to become two.

DNA of ���chromosome

Cell containing gene ���of interest

Gene inserted into���plasmid

Plasmid put into ���bacterial cell

Recombinant���DNA (plasmid)

Recombinant���bacterium

Bacterial ���chromosome

Bacterium

Gene of���interest

Plasmid

1

2

_______ ______ is production of many identical copies of the same gene.

If the inserted gene is replicated and expressed, we can recover the _______________or ________________.

HowIt’sDone

•  Cuts DNA at specific points.•  Cleaves vector (plasmid) and foreign (human) DNA. •  Cleaving DNA makes DNA fragments ending in short single-

stranded segments with “______________”•  These allow for insertion of foreign DNA into vector DNA.

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A

T

A

T

A

T A

T

A

T

C

G

C

CG

G

C

G

A

T

A

T

A

T A

T

A

T

C

GC

CG

G

C

G

Google: McGraw-Hill “Restriction Endonucleases”

Like…

DNA Ligase

•  Job?

•  DNA “_____________” has now occurred

•  The cell membranes of bacteria can be made more permeable to take up plasmids at this point.–  Bacteria and plasmids will

reproduce.–  Many copies of the plasmid and

many copies of the foreign gene are now produced.

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Remember its function in DNA replication?

Google: mcgraw-hill: “Steps in Cloning a gene”

Go to www.sumanasinc.comAnimations-molecular biology-plasmid cloning

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PCR

8  

PCR  cycles  

DNA  copies  

first   1  

second   2  

third   4  

old  

old  

old  strand  

new  

new  

new  strand  

DNA  double  strand  

fourth   8  fi?h   16  and  so  forth  

A three-step cycle—______________, ________________, ____________ —brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

Go to www.sumanasinc.comAnimations-molecular biology-PCR

5ʹ′

Genomic DNA

TECHNIQUE

Cycle 1 ���yields

2molecules

Cycle 2 ���yields

4molecules

Cycle 3 ���yields 8

molecules; ���2 molecules ���

(in white ���boxes) ���

match target ���sequence

Target ���sequence

3ʹ′

3ʹ′

3ʹ′

3ʹ′

5ʹ′

5ʹ′

5ʹ′1

2

3

Advertisement for thermocycler (machine used for PCR)

http://www.cnpg.com/video/flatfiles/539/

Applications of PCR: Analyzing DNA Segments

•  ________ ________________________ is the technique of using DNA fragment lengths called RFLP’s to match/investigate DNA samples.

•  HOW?– By treating DNA samples with restriction

enzymes, a unique collection of different length fragments is produced

– Electrophoresing these fragments separates the fragments according to their•  __________________

– Usually used to measure number of repeats of short sequences

– Produces distinctive banding pattern10

GOOGLE: MCGRAW - HILL “RESTRICTION FRAGMENT LENGTH POLYMORPHISM”

Electrophoresis is a laboratory technique for separating mixtures of charged molecules.

What is Electrophoresis?

•  __________________: a material composed of two or more elements or parts.

• ___________________: a molecule (such as a protein or DNA) that has too many or too few electrons.

Charged molecules are separated based on their electrical charge and size.

Mixture of Charged

Molecules

•  Law Enforcement Agencies

•  Hospitals and Research

•  Paternity Testing

•  Biologists helping animals:

Zoos can use this technique to reduce possibilities of inbreeding. Taxonomists and evolutionary biologists can learn more about evolutionary relationships, identify species and document differences between them. Animals behaviorists can use DNA to glean information about the kinship of animals and how it impacts interactions with other animals

WHO USES IT?

Scientists can use this procedure to examine biological evidence found at crime scenes

Identify damaged genesIdentify viruses and bacterial genomesIdentify genetic diseases such as sickle cell anemia, Huntington’s disease, Duchenne’s muscular dystrophy,

Gene&cists  can  determine  paternity  as  well  as  establishing  gene&c  links  between  large  groups  of  people  

•  ___________________________– the flow of electric charge

•  Acts as a _____________, a device to filter small particles out of a mixture of larger particles.

Essentials parts of GEL ELECTROPHORESIS

How Separation Occurs

Many molecules (amino acids, peptides, proteins, DNA, and RNA) have naturally occurring negative and positive charges on them. The sum of these charges determines the overall charge.

When introduced to an electrical current, negatively charged molecules are attracted to the positive electrode and positively charged molecules are attracted to the negative electrode.

-­‐   +  

-­‐  +  

-­‐  +   -­‐  

+  -­‐  

+  

-­‐   +  

-­‐   -­‐  +  -­‐   -­‐  

-­‐  -­‐  

Nega&vely  Charged  Protein  

-­‐   +  

-­‐  +  +  

Posi&vely  Charged  Pep&de  

+  

N  

N  

O  +  

Posi&vely  Charged                                                                                                                  Amino  Acid  

Molecule Size:The porous material is made of microscopic particles suspended in a gel. The microscopic particles attach to one another forming tunnels that act as a sieve to separate the molecules. Small molecules can move faster than large molecules.

Porous  Material  

How Separation Occurs

Fig.  20-­‐9a  

Mixture  of  DNA  mol-­‐  ecules  of  different  sizes  

Power  source  

Gel  

Anode  Cathode  

TECHNIQUE  

1  

2  

Power  source  

–   +  

+  –  

Homework:  Do  a  virtual  DNA  fingerprin&ng  exercise  GOOGLE:  NOVA  +  CREATE  A  DNA  FINGERPRINT  

Normal���allele

Sickle-cell ���allele

Large ���fragment

Electrophoresis of restriction fragments��� from normal and sickle-cell alleles

201 bp ���175 bp

376 bp

DdeI restriction sites in normal and��� sickle-cell alleles of β-globin gene

Normal β-globin allele

Sickle-cell mutant β-globin allele

DdeI

Large fragment

Large fragment

376 bp

201 bp175 bp

DdeIDdeI

DdeI DdeI DdeI DdeI

Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene

DNA Fingerprinting & Paternity

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Mother   Child   Male  1   Male  2  

Base  re

peat  units  

many  

few  

DNA  Band  paQerns  

Increasing  size  

b.  Automated  DNA  fingerprinTng  

a.  

Fluo

rescen

ce  units  

Who  is  the  father?  Male  1  or  2?  

GOOGLE:MCGRAW-HILL “DNA fingerprinting”

Go to www.sumanasinc.comAnimations-biotechnology-paternity testing

STR’s:  short  tandem  repeats  

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A method of profiling called STR analysis uses loci in DNA where the same sequences of bases may occur a number of times.

For example the sequence GACT may be repeated 5x in one individual and 20x in another individual. The number of repeats varies from person to person

This can help identify individuals from each other or biological evidence

This  is  the  FBI’s  13  Loci  standard  for  matching  individuals  to  evidence.    The  possibility  of  two  individual’s  coding  the  same  in  STR’s  for  the  13  loci  is  greater  than  1  in  a  billion  

Biotechnology Products•  Genetically engineered organisms can produce

biotechnology products. They are called “genetically modified organisms” or (_______’s)

•  Organisms that have had a foreign gene inserted into them are ______________.

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Transgenic Bacteria•  Genes of interest are inserted

into bacteria.•  Bacteria are grown in large vats

called ________________ and product is harvested.–  Products on the market include

insulin, hepatitis B vaccine, t-PA, and human growth hormone.

•  Some genetic modifications can promote the health of plants and other can degrade harmful substances:

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Transgenic Plants•  Agricultural Crops–  Foreign genes now give cotton, corn, and potato strains the

ability to produce an insect toxin–  Soybeans are now resistant to a common herbicide – Weed-killer resisting crops are now common (“Roundup”

ready)

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Human Hormone Production:

Transgenic Animals

•  Vortex Mixing:– Many types of animal eggs

have taken up the gene for bovine growth hormone (bGH) through a procedure that uses agitation, tiny needles and foreign DNA to insert a gene into the egg.

– The procedure has been used to produce larger livestock animals

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microinjection of human gene

.

.

milk

Genes coding for therapeutic & diagnostic proteins are incorporated into an animal’s DNA-The proteins appear in the animal’s

Gene Pharming:-Use of transgenic farm animals to produce pharmaceuticals

Plans are to produce drugs to treat:

Treating Disorders

! __________ ______ involves procedures to give patients healthy genes to make up for a faulty gene.

! It also includes the use of genes to treat genetic disorders and various human illnesses.

! There are __________ (outside body) and __________ (inside body) methods of gene therapy.

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◦  In Vivo◦  Ex Vivo" Children with Severe Combined Immunodeficiency (SCID)

Human Genome Project

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_________________ is the study of genomes of humans and other organisms.

_____________- All the genetic information of an individual (or species)

Goals of Human Genome Project

Did it ever get done? How many base pairs?

•  Humans have between ____________ genes (still being researched)–  Most genes code for proteins–  Much of the other DNA was formerly described as “junk”

•  “Junk” did not specify the order of amino acids in a polypeptide

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Recent observations suggest that between 74% and 93% of the genome is transcribed into RNA but not all translated into proteins

Therefore, the “junk” (nontranslated RNA) may code for regulatory gene expression necessary for proper cellular events.

Gene Structure ! Historically, genes were

defined as discrete units of heredity that corresponded to a locus on a chromosome.

! Prokaryotes typically possess a single circular chromosome without ____________ and have less DNA.

! Eukaryotic chromosomes with much more DNA are much more complex.

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Eukaryotic Gene Structure•  ___________________________ are DNA sequences

that occur between genes•  __________________ DNA elements occur when the

same sequence of two or more nucleotides are repeated many times along the length of one or more chromosomes.

•  __________________ are specific DNA sequences that have the remarkable ability to move within and between chromosomes. AKA: jumping genes!

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Remember STR’s?... Can also be referred to as ______________

YouTube: Transposons: Shifting segments of the genome

•  The study of the structure, function, and interaction of cellular proteins

•  At least 20-25K of our genes are translated into proteins

•  The sum total of these is called the human __________

•  Understanding protein function is essential to the development of better drugs. How?

•  Once the primary structure of these protein is known–  It should be possible to predict their tertiary structure–  Computer modeling of the tertiary of these proteins is an important part of

proteomics

33Worth a look -one last link for biotech products from BIO-RAD:http://bio-rad.cnpg.com/Video/flatFiles/799/

Current understanding of what a “Gene” is…

•  A genomic sequence (either DNA or RNA) directly encoding functional products, either RNA or protein.

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