What is the model for DNA replication?

Semiconservative wins!!

DNA REPLICATION DNA REPLICATION DNA doubles itself in a process called REPLICATION. SIX billion basepairs are copied in each cell.

Steps in replication: 1) DOUBLE STRANDED DNA “UNZIPS” (DNA helicase)

- the hydrogen bonds between A/T and C/G weaken and the DNA splits into two strands.

DNA REPLICATIONDNA REPLICATIONSteps in replication:

2) Special enzymes (polymerases) take nucleotides floating in the cytoplasm and match them to their complimentary nucleotides on the old strand. This makes two double strands each made of an old DNA strand and a new one = SEMICONSERVATIVE REPLICATION.

DNA REPLICATIONDNA REPLICATIONSteps in replication:

2) POLYMERASE III makes a new strand by lining nucleotides up beside each old strand (leading strand – continuous line moves in 5’ to 3’ direction)

POLYMERASE I makes a new strand, but uses short fragments (Okazaki Fragments) to move in 5’ to 3’ direction (lagging strand)

DNA LIGASE joins the Okazaki fragments together.

DNA REPLICATIONDNA REPLICATION

DNA REPLICATIONDNA REPLICATION

DNA REPLICATIONDNA REPLICATIONSteps in replication:

3) ERRORS ARE FIXED.

-         other enzymes check for mistakes in the replication then mistakes are snipped out (by other enzymes) and fixed by Polymerases.

DNA REPLICATIONDNA REPLICATION

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookTOC.html

http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/dna-rna2.swf

3d animations - DNA LEARNING CENTER

SHOW REPLICATION CLIP

DNA Replication

DNA Technology

Read “Witness for the Prosecution”

and answer the questions….. to be

collected on Wednesday.

DNA TechnologyDNA Technology

DNA ligase  In recombinant DNA technology, an enzyme that seals together two DNA fragments from different sources to form a recombinant DNA molecule. (Bacterial recombination clip)

DNA polymerase  In DNA replication, the enzyme that links the complementary nucleotides together to form the newly synthesized strand. PCR – Polymerase Chain Reaction – to amplify amounts of DNA

RESTRICTION ENZYMES – Enzymes that very specifically cut “palindromic” sequences in the DNA. Different restriction enzymes cut in different places making varying sizes of DNA fragments.

DNA TECHNOLOGYDNA TECHNOLOGYWHAT IS A PALINDROME?- A sentence read the same backwards as forwards.

DNA-LAND

MA HAS A HAM

A SANTA AT NASA

NEIL A. SEES ALIEN

RATS LIVE ON NO EVIL STAR

A CAR, A MAN, A MARACA

A MAN, A PLAN, A CANAL, PANAMA

A TOYOTA. RACE FAST, SAFE CAR. A TOYOTA

DNA TECHNOLOGYDNA TECHNOLOGYWHAT IS A PALINDROME?- A sentence read the same backwards as forwards.

SOME MEN INTERPRET NINE MEMOS.

ON A CLOVER, IF ALIVE, ERUPTS A VAST, PURE EVIL, A FIRE VOLCANO.

SUBI DURA A RUDIBUS

DNA TechnologyDNA Technology

DNA ligase  In recombinant DNA technology, an enzyme that seals together two DNA fragments from different sources to form a recombinant DNA molecule. (Bacterial recombination clip)

DNA polymerase  In DNA replication, the enzyme that links the complementary nucleotides together to form the newly synthesized strand. PCR – Polymerase Chain Reaction – to amplify amounts of DNA

RESTRICTION ENZYMES – Enzymes that very specifically cut “palindromic” sequences in the DNA. Different restriction enzymes cut in different places making varying sizes of DNA fragments.

DNA TECHNOLOGYDNA TECHNOLOGYHow do restriction enzymes recognize palindromic DNA sequences?

A

T

C

G

A

T

C

T

T

A

A

G

A restriction enzyme called ECO RI Recognizes and cuts at:

GAATTC CTTAAG

Bacterial Restriction

DNA TECHNOLOGYDNA TECHNOLOGYA Restriction enzyme called ECO RI Recognizes and cuts at:

GAATTC CTTAAG

TTACGAATTCCCATGGTAGAATTCAAACCGTGAATTCA AATGCTTAAGGGTACCATCTTAAGTTTGGCACTTAAGT

THESE CUTS GIVE 4 FRAGMENTS, EACH OF A DIFFERENT SIZE. (IE. 5BP, 14 BP, 13BP, 6BP)

DNA TECHNOLOGYDNA TECHNOLOGYThese fragments, each of a different size. (IE. 5BP, 14 BP, 13BP, 6BP) Can then be separated using GEL ELECTROPHORESIS. DNA Has a negative charge and will move in an electric current. Its ability to go through the gel is limited by its size.

DNA TECHNOLOGYDNA TECHNOLOGYAs small fragments move further and large fragments stay behind, a series of “lines” appears in the gel. Like a barcode, lines can be “read” as a DNA FINGERPRINT. This fingerprint may be different based on the restriction enzyme used.

2 Samples with the same fingerprint may be from the same source. Often, more than one enzyme is used to increase reliability.

http://www.dnalc.org/resources/3d/index.html

Gel electrophoresis simulation

What if we need more DNA?

PCR – polymerase chain reaction

That is the technology answer!!

Another one

- DO LAB 7-8 – DNA FINGERPRINTING (PARTNERS)

- DO FORENSIC SCIENCE LAB

Late one night, the famous rock star, Rockina, returned to her luxurious apartment from an appearance at a concert. As she entered her locked apartment, she noticed that everything in her apartment was a mess the drawers had been emptied out onto the floor; the cushions on the couch were ripped open; and the safe behind the picture on the wall had been opened. She then noticed that the lights were on in her bedroom.

The Case of the Murdered Rock StarThe Case of the Murdered Rock Star

She stormed into the bedroom and surprised a burglar in the process of removing her magnificent (and expensive) jewelry from its hiding place beneath the mattress. Indignant, she jumped on the burglar and tried to stab the person with her nail file. While she was able to inflict a small wound, she was no match for the assailant's knife. In the subsequent struggle, she was killed, and the murderer escaped with her jewelry.

B.The Investigation

When the housekeeper, Casparina, entered her apartment the next day, she saw the body and immediately called the police. When they noted that there had been no signs of a forced entry, the investigation narrowed down to people who knew Rockina and who had a key to enable them to enter her apartment. The suspects were:

1. Casparina, the housekeeper, who had just had a bitter argument with Rockina over a refused raise in salary.

2. Lucifer, her former boyfriend, who she had just jilted for another man.

3. Pinky, the leader of her weight lifting class, who was her new boyfriend. It was rumored that Pinky was insanely jealous of Rockina's fame.

When it was established that all three of the suspects had a key to Rockina's apartment, all had a motive for killing her, and all had no ironclad alibi for the evening that she was killed, the police realized they had a problem. They consequently decided to hire a world famous forensic science team to use DNA fingerprinting to prove which of the suspects was guilty of Rockina's murder.

ECO RI =

FROM THE NAIL FILE

TTGAGAATTCAAGCTTCCGATGGAATTC…AACTCTTAAGTTCGAAGGCTACCTTAAG…

5 18

- Lab 7-8

-Rockina Murder

-The Falcon Murder:

- the evidence sample.

- DNA fingerprint

Karyotype

CYNTHIA and ROY – NOT RULED OUT OF O- OR FLAT FOOTED

LORI, CAROL, AND JOANNE – COULD BE O- AND ARE FLAT FOOTED

XX means it is a girl (not Ray)46 chromosomes (not 47)

means normal (not Down Syndrome) (not Carol)

CYNTHIA– NOT RULED OUT OF O- OR FLAT FOOTED

LORI AND JOANNE – COULD BE O- AND ARE FLAT FOOTED

E. THE CASE RE-OPENS – DNA FINGERPRINTINGOver twenty years, technological advancements made it

possible to re-open the case. DNA fingerprinting was now a reliable and affordable method of analyzing blood evidence. Many of the Falcon family had moved back to Okotoks where they lived off their inheritance. Detectives could now subpoena DNA samples from various members of the family to produce DNA fingerprints. They could also take blood from the knife to amplify and produce a DNA fingerprint. There were still many of the Falcon clan living in the hills. Others, with their wealth, hid themselves behind pricey lawyers. Police were confident, however, that they could use DNA profiling from key family members to obtain warrants to test all of their suspects.

E. THE CASE RE-OPENS – DNA FINGERPRINTINGYou are going to use the restriction enzyme, ECORI, to create a DNA

fingerprint of key people and of the blood evidence from the knife. ECORI has the following recognition sequence:

G AATTC the cut is between the G and A.C TTAAG

PROCEDURE:- FIND and MARK cut sites on all the strands of DNA.- COUNT the size of each restriction fragment in base pairs (count on

the top row of DNA)- On the basis of fragment size, draw corresponding lines in the lanes on

the gel for each of the family members and for the evidence.- INTERPRET the DNA fingerprint to ELIMINATE suspects and make

guesses about the identity of the killer (in the box beside your gel).

(E) CONSTRUCT A DNA FINGERPRINTSTEP 1: CUT WITH ECO R1

- COUNT THE SIZE OF PIECES

THE KNIFECCCGAGAATTCAAGCTTAGGCTAGAATTCAGGCTACGAATTCCTGAATTCTAGGCTATAGGCAGGCTACGAATTCGGAGGCTATGGGGCTCTTAAGTTCG AATCCGATCTTAAGTCGGATGCTTAAGGACTTAAGATCCGATATCCGTCCGATGCTTAAGCCTCCGATAC

BOBBYCCGAATTCTCGAATTCTAGGCTATAGGCAGGCTACGAATTCGGAGGCTATGAATTCCGAGTGAATTCGGAGGCTACCCATCCGATGGCTTAAGAGCTTAAGATCCGATATCCGTCCGATGCTTAAGCCTCCGATACTTAAGGCTCACTTAAGCCTCCGATGGGTAGGCTA

NINACCCGAATTCGAATTCAGGCTAAGGCTAGAATTCAGGCTACGAATTCTCGGAGGCTAAAAGCCCGAATTCGCAAATCATGGGCAGCGGGCTTAAGCTTAAGTCCGATTCCGATCTTAAGTCCGATGCTTAAGAGCCTCCGA TTT TCGGGC TTAAGCGTTTAGTACCCGTCG

CYNTHIACGAATTCAGGCTATCCGAGATGAATTCAGGCTACGAATTCTTAGGCTACCCACAGGCTACCGAATTCGAAGACAGGCTAGAATTCGCTTAAGTCCGATAGGCTCTAC TTAAGTCCGATGCTTAAGAATCCGATGGGTGTCCGATGGCTTAAGCTT CTGTCCGA TCTTAAG

LYNNECCCGACAATTCAGGCTAAGGCTAGAATTCAGGCTACGAATTCCTGAATTCTAGGCTATAGGCAAGCGAATTCTTGAATTCCTATGGGGCTGTTAAGTCCG ATTCCGATCTTAAGTCCGATGCTTAAGGACTTAAGATCCGATATCCGTTCGCTTAAGAACTTAAGGATAC

GAATTC CTTAAG

(E) CONSTRUCT A DNA FINGERPRINTSTEP 2: RUN THE DNA PIECES ON A GEL

THE KNIFE 6, 18, 13, 8, 25, 15CCCGAGAATTCAAGCTTAGGCTAGAATTCAGGCTACGAATTCCTGAATTCTAGGCTATAGGCAGGCTACG AATTCGGAGGCTATGGGGCTCTTAAGTTCG AATCCGATCTTAAGTCGGATGCTTAAGGACTTAAGATCCGATATCCGTCCGATGCTTAAGCCTCCGATAC

BOBBY 3, 8, 25, 15, 11, 23CCGAATTCTCGAATTCTAGGCTATAGGCAGGCTACGAATTCGGAGGCTATGAATTCCGAGTGAATTCGGAGGCTACCCATCCGATGGCTTAAGAGCTTAAGATCCGATATCCGTCCGATGCTTAAGCCTCCGATACTTAAGGCTCACTTAAGCCTCCGATGGGTAGGCTA

NINA 4, 6, 18, 13, 23, 21CCCGAATTCGAATTCAGGCTAAGGCTAGAATTCAGGCTACGAATTCTCGGAGGCTAAAAGCCCGAATTCGCAAATCATGGGCAGCGGGCTTAAGCTTAAGTCCGATTCCGATCTTAAGTCCGATGCTTAAGAGCCTCCGA TTT TCGGGCTTAAGCG TTTAGTACCCGTCG

CYNTHIA 2, 20, 13, 27, 18, 5CGAATTCAGGCTATCCGAGATGAATTCAGGCTACGAATTCTTAGGCTACCCACAGGCTACCGAATTCGAAGACAGGCTAGAATTCGCTTAAGTCCGATAGGCTCTAC TTAAGTCCGATGCTTAAGAATCCGATGGGTGTCCGATGGCTTAAGCTT CTGTCCGA TCTTAAG

LYNNE 24, 13, 8, 22, 8, 10CCCGACAATTCAGGCTAAGGCTAGAATTCAGGCTACGAATTCCTGAATTCTAGGCTATAGGCAAGCGAATTCTTGAATTCCTATGGGGCTGTTAAGTCCG ATTCCGATCTTAAGTCCGATGCTTAAGGACTTAAGATCCGATATCCGTTCGCTTAAGAACTTAAGGATAC

(E) CONSTRUCT A DNA FINGERPRINT

(E) CONSTRUCT A DNA FINGERPRINT

LORI AND JOANNE – COULD BE O- AND ARE FLAT FOOTED;ARE DAUGHTERS OF DENNY AND NINA. THE KILLER COULD BE DENNY AND NINA’S CHILD.