Molecular Bio 2

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hybridization:

Back 1

complementarity allows two fragments of DNA to hybridize

anealing of a DNA probe to its target sequence

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southern hybridization

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hybridization of a probe fragment to a DNA target

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northern hybridization

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hybridization of a probe fragment to a RNA target

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PCR

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polymerase chain reaction:
based on primer complementarity and repetitive synthesis...
requirement: template, pair of primers four deoxyribonucleotides
Taq DNA polymearse
appropriate buffer

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microarray/chip based on hybridization

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determining differential expression of genes in two tissues or cell types by using a pool of probes

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DNA applications include what that are important to medicine and mentioned in his slide?

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disease diagnosis
prenatal diagnosis
bacterial infections
virus infection and viral load

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Forensice medicine

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paternity test
innocent vs guilty verdict

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statistical analyses

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DNA analyses in forensics involves rigorous analyses

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VNTR

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variable number of tandem repeats

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medical applications of molecular differences of DNA replication between prokaryotes and eukaryotes

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anitbiotics and such

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cell doctrine

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every cell in the body arises form an existing cell

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cell cycle

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orderly sequence of DNA duplication and cell division

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cell cycle is controlled by what?

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cyclins and cyclin dependent kinases

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What are the checkpoint of the cell cycle?

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1. DNA replicates only when conditions are right
2. Mitosis occurs only after replication has completed
3. cytokinesis occurs only after all chromosomes have separated

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semiconservative:

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each stand serves as template to specify its complementary strand

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what enzyme carries out DNA replication in e. coli?

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DNA polymerase

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What are the three types of e. coli polymerases?

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Polymerase I - DNA repair
Polymerase II- DNA repair
Polymerase III- DNA replication

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what are the activities of pol I

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5'-3' polymerization
5'-3' exonuclease
3'-5' exonuclease

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What genes code for Pol I, II, and III?

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polA
polB
dnaE

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What does dnaA, dnaB and dnaG code for?

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dnaA-initiator protein
dnaB-helicase
dnaG-primase

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what happens when a wrong base is incorporated?

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DNA polymerase has an editing (exonuclease) activity

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e. coli has what kind of genome?

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double stranded circular genome

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Where does replication begin?

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origin of replication...a defined sequence

initiator protein (dnaA) binds to the origin an denatures A-T base pairs

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What enzyme acts after initiator protein?

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helicase (dnaB) ...the double stranded DNA will be unwound...dependent on ATP

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What stabilizes the unwound DNA strands?

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single strand binding protein

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Unwinding of one part of DNA leads to overwinding of other parts of DNA. What relieves the overwinding?

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type II topoisomerase (gyrase in e. coli)

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What comes next after DNA is unwound?

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primase synthesiszes 15 nt long RNA primers

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_____ adds new nucleotides to the 3'-OH end of the growing chain.

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DNA polymerase III

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What enzyme acts after DNA polymearse III?

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DNA polymerase I
-removes RNA primers
-RNA primers are replaced by extending DNA synthesis

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What enzyme acts after DNA polymerase I?

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DNA ligase...it seals the gap
5'P to 3'-OH

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e. coli polymerase III (holoenzyme)

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has several subunits
two catalytic cores of three subunits (alpha, epsilon, theta) one for leading and one for lagging strand
clamp subunits (beta, beta)
clamp loader (gamma complex)
assembly subunit (t, t)

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If we were to incubate in a test tube: dAP, dGTP…and buffer at appropriate temerature. Will a polymer be made?

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no, need a templete

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supercoiling

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positive super coiling (overwound)

negative supercoiling (underwound)

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gyrase

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heterotetramer

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what is the target enzyme for anticancer drugs?

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mammalian topo I

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topoisomerase I

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makes a cut on one strand of the DNA and binds to 3' end of phosphodiester back bone

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topotecan (hycamtin)

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FDA approved for ovarian cancer and small cell lung cancer

stabilized topo I-DNA complex and prevent sthe religation step