DNA Replication

Question 1

Nucleotide Structure and Uses?

Answer 1

Phosphate Grp, Sugar (ribose/deoxyribose), Nitrogenous base (A, T, C, G, U)
Uses: store energy, monomer or nucleic acid, co-enzymes

Question 2

Difference btw purines and pyrimidines?

Answer 2

Purines - Dbl ring
Adenosine (NH2)
Guanosine (NH2, dbl bonded O)
Pyrimidines - Single ring
Cytosine (NH2, dbl bonded O)
Thymine (CH3, dbl bonded O)
Uracil (2 dbl bonded O)

Question 3

What sugars apart of nucleic acids?

Answer 3

Ribose - OH on C#2
Deoxyribose - missing O from C#2
*both pentose

Question 4

What are abbreviations? ie. dNTP

Answer 4

d - deoxyribose, no “d” - ribose
N - base, (A, T, U, C, G)
T - # of phosphates, M=mono, D=di, T=tri

Question 5

How to nucleotides link together?

Answer 5

Phospodiester linkage between sugar of one and phosphate of another

Question 6

Structure of DNA?

Answer 6

Dbl helix - hydrogen bonding
Strands antiparallel
Long strands

Question 7

Base Pairing?

Answer 7

A with T (2 H bonds)

C with G (3 H bonds)

Question 8

Ribonucleic Acid (difference btw DNA)?

Answer 8

Shorter, ss
Found in and outside nucleus (DNA only in)
Different base (U) and sugar (ribose)

Question 9

When does DNA replicate?

Answer 9

• everytime cell divides
• S phase of interphase
• complete set of identical chromosomes must go into daughter cell

Question 10

DNA semi-conversative when replicating?

Answer 10

• use parent strand as copy to produce two identical molecules
• each synthesized strand composed of one newly synthesized and parent strand
• requires enzymes and is an endergonic process

Question 11

What is DNA gyrase?

Answer 11

• relieves tension caused by the unwinding of the dbl helix

- bacterial enzyme

Question 12

What is helicase?

Answer 12

-breaks H bonds btw nucleotides (unzipping molecule)

Question 13

What are ssB’s?

Answer 13

• single stranded binding proteins

- facilitate unwinding of DNA molecule

Question 14

What does primase do?

Answer 14

• lays down RNA primers to build complementary strands

* free floating nucleotides can then base pair with nucleotides alond ssDNA

Question 15

What is DNA polymerase?

Answer 15

• works 5’ to 3’ and builds new strand of DNA by attaching nucleotides
• nucleotides lose 2 phosphates to generate E for process
• replaces RNA primers with DNA nucleotides
• new strand formation happens in opposite directions along DNA

Question 16

What does ligase do?

Answer 16

-joins gaps btw Okazaki fragments on lagging strand

Question 17

Difference btw lead/lag strand

Answer 17

1. Lead - 3’ to 5’, replication continuous towards replication fork
2. Lag - 5’ to 3’ replication discontinuous away from replication fork, in Okazaki fragments

Question 18

Define: replication fork & replication bubble

Answer 18

replication fork - region where enzymes replicating DNA untwist molecule
replication bubble - region where two replication forks are close in proximitiy

Question 19

Why to cells die?

Answer 19

• everytime DNA molecule replicated, primer on ends not replaced or replicated results in lose of approx 100 bp.
• telomeres are non-coding DNA sequence at end of molecule, protect important genetic info from being eroded
• DNA gets progressively shorter
• once telomeres completely eroded important info starts to erode cells die
• telomerase extends telomeres

Question 20

What are two genes that regulate mitosis?

Answer 20

-when mutation occurs problem arises as unblances them
oncogenes: increase mitosis
tumor suppressor genes: decress mitosis

Question 21

3 changes that happen to cancer cells?

Answer 21

1. immortalization: cells don’t die
2. transformation: cells don’t function normally
3. metastisis: cells become mobile
   * cancer cells have large quantites of telomerase

Question 22

What is proof reading?

Answer 22

• as DNA polymerase replicates proof reads
• only can change right then, once nucleotide placed cannot fix
• 99% accurate (lots of error)

Question 23

What is exicision repair?

Answer 23

• bacterial enzymes that recongnize mistakes in DNA, cut out sequence
• DNA polymerase repairs

Question 24

What are restriction enzymes?

Answer 24

• bacterial enzymes that cut out specific sequence (recognition site) of DNA
• sequence palindromic 4-6 bp

Question 25

Blunt Ends

Answer 25

-cut straigt through molecule

Question 26

Sticky Ends

Answer 26

-staggered cuts through molecule, produce some ssDNA

Question 27

What is PCR?

Answer 27

- way to mass replicate DNA using one single molecule | - involves exponential copying of DNA

Question 28

Steps for PCR

Answer 28

1. Mix in vial DNA, primers, nucleotides, DNA polymerase 2. Heat mixture to denature molecule (90), cool temperature (60) 3. Anneal primer to appropriate ends of ssDNA 4. Nucleotides base pair to complementary strand using Taq polymerase

Question 29

Why does polymerase not have to work in 3' to 5'?

Answer 29

-manufactured

Question 30

How many times is PCR repeated?

Answer 30

- 20 to 30 times | - used to clone genes for mass production

Question 31

What % of DNA is identical/different?

Answer 31

99% identical, 1% different

Question 32

Steps of DNA fingerprinting.

Answer 32

1. Stain DNA 2. Place DNA in well with gel (agarose) 3. Attach unit to power source 4. DNA will migrate to positive terminal since its negatively charged 5. Small fragments move faster than large

Question 33

What is DNA recombination and its applications?

Answer 33

-incorporating a gene into an already existing genome | Applications: GMOs, pharmacology, agriculture, gene therapy

Question 34

What is viral reproduction?

Answer 34

- viral DNA inserted into bacteria cell, adding viruses genes to host cell - cell expresses genes - bacteria share genetic info through passing of plasmids back and forth - good vectors

Question 35

What is a vector?

Answer 35

-agents that carry genes into cells

Question 36

Different parts of Plasmid.

Answer 36

ori- origin of replication- re- restricted sites ar- antibiotic resistance

Question 37

What are steps of producing recombinant DNA?

Answer 37

1. Cut plasmid - using restriction enzymes 2. Mix in desired genes - with same ends 3. Attach gene to plasmid - match sticky ends together and splice in gene (promoter at front and antibiotic resistant gene) 4. Some plasmids express gene some don't 5. Put plasmid into bacteria cell - treat with heat/chemicals to force uptake of foreign DNA 6. Bacteria expresses newly added genes

Question 38

What is a promoter?

Answer 38

-located at front of gene, activates gene "turning it on"

Question 39

Explain how compotent cells chosen.

Answer 39

- mixed with antibiotics, bacteria that picked up gene will have antibiotic resistance gene and thrive in presence - host cell produce protein from gene - bacteria who do not express gene will die

Question 40

What is terminology for Sanger Method?

Answer 40

NTP - nucleotide used in ATP/RNA dNTP- deoxynucleotide, used in normal DNA replication ddNTP - dideoxynucleotide, modified missing OH from C#2, not normally used in DNA replication

Question 41

How do you sequence DNA?

Answer 41

Combine - ssDNA, one ddNTP, dNTP, primers and DNA polymerase - anneal primer to template strand and allow to be copied - at one point ddNTP will be incorporated into strand and result in dead end as missing OH from C#2 - procedure repeated with different ddNTP to produce different sized fragments of DNA - run DNA through gel to seperate out segments - read base squence (bottom to top) , write out complementary strand and then original strand