Bioengineering Biology Ch.13 Ms. Haut. 13-2 Manipulating DNA Copyright Pearson Prentice Hall

Bioengineering

Biology

Ch.13

Ms. Haut

The Tools of Molecular Biology

• Scientists use their knowledge of the structure of DNA and its chemical properties to study and change DNA molecules.

Copyright Pearson Prentice Hall



• Scientists use different techniques to:– extract DNA from cells– cut DNA into smaller pieces– identify the sequence of bases in a DNA

molecule– make unlimited copies of DNA



• In genetic engineering, biologists make changes in the DNA code of a living organism.



• DNA Extraction– DNA can be extracted from most cells by a

simple chemical procedure.– The cells are opened and the DNA is

separated from the other cell parts.



• Cutting DNA – Most DNA molecules are too large to be

analyzed, so biologists cut them into smaller fragments using restriction enzymes.

• Enzymes found in bacteria used to destroy phage DNA


• Each restriction enzyme cuts DNA at a specific sequence of nucleotides.


• A restriction enzyme will cut a DNA sequence only if it matches the sequence precisely.

The Tools of Molecular Biology• Separating DNA

– In gel electrophoresis, DNA fragments are placed at one end of a porous gel, and an electric voltage is applied to the gel.

– When the power is turned on, the negatively-charged DNA molecules move toward the positive end of the gel.

BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by Campbell, Reece, Mitchell, and Taylor, ©2003.



• Gel electrophoresis can be used to compare the genomes of different organisms or different individuals.

• It can also be used to locate and identify one particular gene in an individual's genome.


• DNA Fingerprinting– A method of

developing a person’s DNA “profile,” similar to a fingerprint.

– Pioneered in England in 1984 by Dr. Alec Jeffreys

Dr. Alec Jeffreys

First Forensic Use

• First used by law enforcement in England in the mid-1980’s.

• DNA evidence exonerated one man, and convicted another.

• Described in The Blooding, by Joseph Wambaugh

http://www.amazon.com/exec/obidos/tg/detail/-/0553282816/ref=lib_rd_next_2/103-1421887-1011802?v=glance&s=books&vi=reader&img=2

How does it work?

• 99.9% of your DNA is the same as everyone else’s.

• The 0.1% that differs are a combination of:– Gene differences (Differences in the genes

themselves)– Differences in “polymorphic regions” between

the genes on the DNA.

How does it work?

• Certain points between the genes on the DNA have repeating base sequences.– For example:

ATTACGCGCGCGCGCGCGCTAGC– These are called variable number tandem

repeats (VNTRs for short)

How does it work?

• Everyone has VNTRs at the same place in their DNA, but they are different lengths for different people.– For example:

Person 1: ATTACGCGCGCGCGCGCGTAGC(7 repeats)

Person 2: ATTACGCGCGCGCGTAGC(5 repeats)

To Make a DNA Fingerprint…

• First, we use restriction enzymes to chop the DNA up into millions of fragments of various lengths.– Some of the fragments contain STRs;

some do not. The ones that do are different lengths for different people.


• There are slight differences in DNA sequences as seen in individuals of the same species

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• Next, we use gel electrophoresis to sort the DNA fragments by size.

Gel Electrophoresis

• Method for sorting proteins or nucleic acids on the basis of their electric charge and size

Gel Electrophoresis

• Electrical current carries negatively-charged DNA through gel towards positive electrode

• Agarose gel sieves DNA fragments according to size

– Small fragments move farther than large fragments


Gel Electrophoresis



• Finally, a radioactive probe attaches to our VNTRs. Only the fragments with our VNTRs will show up on the gel.

Figure 12.11C

Restriction fragmentpreparation

1

Restrictionfragments

Gel electrophoresis2

Blotting3 Filter paper

Probe

Radioactive probe4

Detection of radioactivity(autoradiography)

5

Film

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http://www.dnalc.org/resources/animations/index.html


• Since VNTRS are different lengths in different people, this creates a DNA Fingerprint.

Two uses for DNA Fingerprints...

• ForensicsDNA taken from crime scenes (blood, semen, hair, etc.) can be compared to the DNA of suspects.

Real-life CSI!


• ForensicsThis is an example of a gel that might be used to convict a rape suspect. Compare the “Sperm DNA” to the “Suspect DNA.” Which suspect committed the rape?


• Paternity TestingSince all of our DNA markers came from either mommy or daddy, we can use DNA fingerprints to determine whether a child and alleged father are related…just like on Maury Povich!


• Look at the two “Child” markers on this gel. Can they both be matched up to either the mother or the “alleged father?”

• Yes. This is a “positive” test for paternity.


• How about this gel? Do both of the child’s markers match either the mother or the “alleged father.”

• No! The “alleged father” is not this child’s biological parent.

Interpreting DNA Fingerprints

• Which child is not related to the mother?

• Son 2

• Which children are not related to the father?

• Daughter 2 and Son 2


• A blood stain was found at a murder scene. The blood belongs to which of the seven possible suspects?

Suspect 3


• Who committed this sexual assault?

Suspect 1


• These DNA fingerprints are from a mother, a child, and two possible biological fathers. Which one is the daddy?

2nd alleged father


• Mother, father, and four children. Which child is from a different father?

Child 2

Using the DNA Sequence

• Cutting and Pasting – Short sequences of

DNA can be assembled using DNA synthesizers.

– “Synthetic” sequences can be joined to “natural” sequences using enzymes that splice DNA together.


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• These enzymes also make it possible to take a gene from one organism and attach it to the DNA of another organism.

• Such DNA molecules are sometimes called recombinant DNA.


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• Making Copies – Polymerase chain reaction (PCR) is a

technique that allows biologists to make copies of genes.

– A biologist adds short pieces of DNA that are complementary to portions of the sequence.

http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html


• DNA is heated to separate its two strands, then cooled to allow the primers to bind to single-stranded DNA.

• DNA polymerase starts making copies of the region between the primers.


Genomic DNA

Targetsequence

5

3

3

5

5

3

3

5

Primers

Denaturation:Heat brieflyto separate DNAstrands

Annealing:Cool to allowprimers to formhydrogen bondswith ends oftarget sequence

Extension:DNA polymeraseadds nucleotides tothe 3 end of eachprimer

Cycle 1yields

2molecules

Newnucleo-

tides

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

Genomic DNA

Targetsequence

5

3

3

5

5

3

3

5

Primers

Denaturation:Heat brieflyto separate DNAstrands

Annealing:Cool to allowprimers to formhydrogen bondswith ends oftarget sequence

Extension:DNA polymeraseadds nucleotides tothe 3 end of eachprimer

Cycle 1yields

2molecules

Newnucleo-

tides

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

http://www.dnalc.org/resources/animations/pcr.html


Restriction enzymes are used toa) extract DNA.

b) cut DNA.

c) separate DNA.

d) replicate DNA.


During gel electrophoresis, the smaller the DNA fragment is, the

a) more slowly it moves.

b) heavier it is.

c) more quickly it moves.

d) darker it stains.


The DNA polymerase enzyme Kary Mullis found in bacteria living in the hot springs of Yellowstone National Park illustrates

a) genetic engineering.b) the importance of biodiversity to biotechnology.c) the polymerase chain reaction.d) selective breeding.


A particular restriction enzyme is used toa) cut up DNA in random locations.

b) cut DNA at a specific nucleotide sequence.

c) extract DNA from cells.

d) separate negatively charged DNA molecules.


During gel electrophoresis, DNA fragments become separated because

a) multiple copies of DNA are made.

b) recombinant DNA is formed.

c) DNA molecules are negatively charged.

d) smaller DNA molecules move faster than larger fragments.


Why would someone use PCR?a) To cut the DNA into fragments

b) To create recombinant

c) To make lots of copies of a DNA fragment

d) To make a transgenic organism

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Bioengineering Biology Ch.13 Ms. Haut. 13-2 Manipulating DNA Copyright Pearson Prentice Hall