Biochem Exam 2

DNA bands are visualized by staining with

ethidium bromide which is florescent when bound to DNA (changes the dialetric, decreases homolumo gap)

DNA can be purified based on

size and charge using an agarose gel electrophoresis

what are the role of endonucleases?

they break the phosphate backbone of DNA.

DNA restriction endonucleases recognize

specific sequences to bind and cleabe the phosphate backbone

How do endonucleases recognize specific DNA sequences?

through sidechain H-bonds

Endoncleases cut their specific sequence by stabalizing a ______ ______ ______ ____, allowing water to cleave the phosphate backbone

pentavalent phosphate transition state

what is used to cut and paste DNA sequences into plasmids

Endonucleases and DNA ligas

How can we amplify the plasmids?

E.coli and purified to obtain mad DNA

sticky ends

created by staggered cuts, leaving short, single-stranded overhangs that can base-pair with complementary sequences.

GGT[G]

CCA[CTTAA]

blunt end

Blunt ends are created by a straight cut, with both DNA strands ending at the same point and having no overhangs

DNA polymerase 

an enzyme that synthesizes new DNA strands from existing ones by adding nucleotides

the main DNA replicative enzyme, responsible for high-speed synthesis of new DNA strands, particularly the leading strand

DNA Polymerase (Pol) III

primarily removes RNA primers from the lagging strand and fills the resulting gaps with DNA, a process involving its unique 5' to 3' exonuclease activity

DNA Polymerase (Pol) I

Primary method for determining the sequence of DNA fragments

DNA sequencing

Primers are elongated by ___ ____ with a nucleotide mixture with 4 ____ ______

DNA polymerase, fluorescent ddNTP

why does ddNTP terminate the DNA sequencing

it lacks an OH on the 3’ carbon

for DNA sequencing, after the the sequence is terminated with ddNTP, the mixture is seperated by

gel electrophoresis and analyzed by laser-excited fluorescence

primer

a short, single-stranded DNA fragment that serves as a starting point for DNA synthesis in laboratory techniques like the polymerase chain reaction (PCR) and DNA sequencing

DNA sequencing steps

1. template strand of unknown sequence

2. generate MAD copies

3. add primers

4. DNA polymerase will synthesize the template strand until the ddNTP stops the sequencing (dye labeled)

5. denature till we just have the complementary strand

6. dye-labeled segments applied to a capillary gel and subjected to gel electrophoresis

7. DNA migrates smallest on the bottom to, largest on the top, 5’ to 3’The 

8. computer generates the results of the complementary strand

9. Template strand is 3’ to 5’ of that strand

in DNA sequencing, the smallest complementary strand is the

5’ end

the number of genes DOES not scale with

genome size and organism complexitythe

the vast majority of human DNA does not code for

functional RNA or proteins

How can we amplify DNA?

through polymerase chain reaction (PCR)