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Biochemistry > DNA Replication > Flashcards

DNA Replication Flashcards

1
Q

What is the central dogma?

A

DNA to DNA Replication

DNA to RNA Transcription

RNA to Protein Translation

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2
Q

What is reverse transcriptase?

A

It is a exception to the central dogma where the RNA genome of retroviruses is converted to DNA. This DNA then is used to make new RNA molecules by the virus infiltrating the host genome.

RNA to DNA to RNA

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3
Q

What is an example of a _________ that uses reverse transcriptase?

A

Retrovirus

HIV

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4
Q

What is RNA dependent RNA polymerase?

A

RNA genome of RNA virus is copied to new molecules of RNA

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5
Q

What is an example of a _____ that utilizes RNA dependent RNA polymerases?

A

virus

COVID

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6
Q

Nucleoside is made of :
Nucleotide is made of:

A

purine/pyrimidine nitrogenous base and pentose ribose/deoxyribose sugar

Nucleoside + phosphates

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7
Q

DNA is a polymer of _____ (#) ___________.

The purines are :
The pyrimidines are:

A

4 deoxyribonucleotides

Adenine, Guanine
Cytosine, Thymine

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8
Q

RNA is a polymer of _____ (#) ___________.

The purines are :
The pyrimidines are:

A

4 ribonucleotides

Adenine, Guanine
Cytosine, Uracil

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9
Q

Purines are made of _______ ring(s) and include ____ and ___.

Which one has only one NH2 group ?

A

2 rings
Adenine and Guanine

Adenine

Rings as PURE As Gold

Rings - 2 Rings
Pure- Purine
As Gold - Adenine and Guanine

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10
Q

Purines are made of _______ ring(s) and include ____ and ___.

Which one is associated with RNA?

A

1 rings

Thymine, Uracil, Cytosine

Uracil

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11
Q

What are the precursors for DNA or RNA synthesis?

A

Triphosphates (dNTPs or NTP)

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12
Q

____________ are incorporated into DNA or RNA and ______ is released.

A

Monophosphates

PPi (pyrophosphate)

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13
Q

DNA and RNA is formed by linking ____ by ________. It grows in the ___ to ___ direction.

Describe it.

A

Nucleotides , Phosphodiester bond

5’ to 3’

The 5’ phosphate group on one nucleotide is joined to the 3’ hydroxyl group of the pentose sugar of the prior nucleotide.

This releases PPi

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14
Q

RNA is hydrolyzed rapidly under ___________ conditions because of the __________ on the _________. DNA is _____ in such conditions.

A

alkaline

2’ OH on the pentose sugar

stable

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15
Q

On DNA, the 2’ carbon on the _______ contains a ______ while on RNA a ______ is present.

A

pentose sugar

H, OH

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16
Q

1’ carbon pentose sugar
2’ carbon pentose sugar
3’ carbon pentose sugar
4’ carbon pentose sugar
5’ carbon pentose sugar

A

1.Holds the purine or pyrimidines

2: H or OH to distinguish RNA vs DNA

3: Participates in phosphodiester bond linkage as nucleotide be added TO. (OH)

4: Provides molecules stability and hosts 5’ carbon

5: Participates in phosphodiester bond linkage as adding nucleotide by using phosphate group.

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17
Q

Adenine and _______ form _______ bonds

Guanine and _______ form ______ bonds

A

Adenine and Thymine form two hydrogen bonds

Guanine and Cytosine form three hydrogen bonds.

GC3 rhymes hehe

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18
Q

The DNA structure is ___________ wound around each other to form ___________. Each “turn” is made of __________.

Coiling of the DNA creates ____________.

A

two sugar phosphate backbones wound around each other to form a right-hand double helix.

Each turn is made of 10.4 nucleotides.

Major groove and minor groove

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19
Q

DNA replication is _______________. This means that the parental DNA double helix will __________.

A

Semi-conserved and discontinuous

serve as template strands for two new complementary DNA strands.

Each new replicated piece has one old and and one new piece of DNA.

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20
Q

DNA must be ____ and both the _________ and ________ copied using ________.

DNA can only synthesize in _______.

A

unwound

5’-3’ and 3’-5’
DNA polymerase

5’-3’

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21
Q

_______ is the assembly of enzymes that replicate DNA. These enzymes are______.

In bacteria ________ proteins are involved in the _____.

A

Repliosome

Helicase
DNA Polymerase
DNA primase.

20-25 , repliosome

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22
Q

ALL DNA polymerase _________________.

A

Synthesize 5’-3’
Require a template
Require a primer with a 3’ OH
Use dNTPs as energy source
Incorporate dNMPS into DNA

23
Q

The ________ strand is continuous while the _______ is not.

A

Leading, Lagging

24
Q

Why is the lagging strand replication of DNA discontinuous?

How is the discontinuity addressed?

A

The lagging strand is the “5’-3’” strand of DNA. When the replication fork opens up in the 5’-3’ direction, DNA polymerase will run into the 5’ end of the DNA strand if it proceeds to add nucleotides in a 5’-3’ fashion (aka the reverse direction of the OG 5’-3’ DNA strand).

Instead, DNA is synthesized in small fragments called Okazaki fragments that are eventually synthesized together to create one long lagging strand.

25
Q

DNA Replication Steps

  1. _________ unwinds DNA ahead of the _______ and __________ straightens DNA template and _________.
  2. The ______ is synthesized in the 5’-3’ direction by _________. Only one _____ is needed at the start of replication for the ________.
  3. ______ are synthesized on the ________ by ________.
  4. ____ are elongated by _______ in the lagging strand. ______ are formed.
  5. _______ is removed by a _______.
  6. Another molecule of ________ fills the gaps between Okazaki fragments where the _________ was but still leaves a _______.
  7. ______ joined the nick between Okazaki fragments.
  8. ____ prevents ______.
A
  1. DNA helicase unwinds DNA double helix ahead of the replication fork

SSB proteins (Single Strand Binding) straightens out DNA template and facilities DNA polymerase

  1. leading strand , DNA polymerase , RNA primer, leading strand
  2. RNA primers are synthesized on the lagging strand by DNA primase.
  3. RNA primers, DNA polymerase. Okazaki fragments
  4. RNA primer is removed by a 5’-3’ exonuclease (RNAse H)
  5. DNA polymerase, RNA primer, nick
  6. DNA ligase
  7. DNA topoisomerase prevents DNA tangling
26
Q

What is DNA Primase?

It does NOT require _____ and can join ________.

A

DNA primase creates short RNA molecules complementary to the DNA template in the 5’-3’ direction in intervals of about 100-200 nucleotides along the lagging strand.

It does NOT require a primer and can join two nucleoside triphosphates.

27
Q

RNAseH is responsible for _________.

A

Exonuclease

Removing RNA primer from lagging strand.

28
Q

DNA topoisomerase is responsible for __________.

A

preventing the tangling of DNA

29
Q

What is the origin of replication?

A

The position in the nucleotide sequence where the DNA helix is opened. A replication bubble is formed.

30
Q

Prokaryotic organisms have ___________ origin of replication while eukaryotic organisms have ______.

A

1 / many

31
Q

Why do eukaryotic organisms have so many origins of replication?

A

Eukaryotic organisms have multiple origins of replication to allow for the synthesis of the complete genome in a reasonable amount of time

32
Q

Replication origins are activated in clusters called ____________.

Replication units are activated at different times during the ________of the cell cycle. Within one replication unit origins are spaced ________ based pairs apart.

A

replication units

S-phase

30K-250K

33
Q

If you periodic cells have multiple origins of replication, how does the replication

A

Individual replication forks proceed from each origin stopping when they collide with a replication fork from the opposite direction.

34
Q

List the following processes from most prone to error to least.

A

5’-3’ polymerization
3’-5’ exonucleolytic proofreading
+Strand-directed mismatch repair

35
Q

Analysis of DNA can be done by_________

A

Sequencing

PCR/qpcr

SNP arrays
Genome Sequencing

36
Q

What can analysis of RNA be done by and how?

A

Reverse transcriptase-PCR / RT-qpcr.

In RNA the expression of a particular gene is used to analyze specific molecules.

RNA-seq

37
Q

What can analysis of proteins be done by and how?

A

Western Blotting and Immunohistochemistry

Is the protein expressed?

Proteomic

38
Q

Describe Sanger Sequencing

A

Sing DNA sequencing, involves en somatic DNA synthesis, using base specific chain terminators and a radio labeled primer.

The base specific chain terminators will terminate at a at C at G and T in four separate rounds.

The DNA products are then denatured on large poly acrylamide gels that can resolve sizes of these products by one nucleotide this will give us different sizes of products ordered from smallest at the end of the polyacrylamide gel to largest at the top, by which we can order the bases of the nucleotides.

Sanger sequencing determines nucleotide sequence in a linear piece of DNA.

39
Q

What are Dideoxy bases?

A

Dideoxy bases are kind of altered nucleotide used in Sanger sequencing

They do not contain a hydroxyl group on the three prime carbon of the pentose sugar which terminates the DNA sequence synthesis.

40
Q

Automated DNA sequencing with fluorescent primers

A

The concept is similar to Sang sequencing, except the four reactions of each nucleotide contain the same primer with a different fluorescent label. Reactions are then combined at once prior to separation, and the signal is detected by a laser.

Gels not needed!

41
Q

PCR

What is the product?

A

PCR is a cycle of heating and cooling DNA strands to replicate a specific region of DNA using an upstream and downstream primer (for the two strands)

The PCR product is the size of the primers and base pairs in between.

42
Q

When is the defined PCR product made?

A

Cycle 2

43
Q

Since PCR involves high temperatures, what molecule is involved in the process that must withstand it?

A

DNA polymerase developed from bacteria in hot springs in Yellowstone

44
Q

Restriction Fragment Length Polymorphisms RFLP analysis

A

If a mutation of a DNA changes the restriction site, you can PCR the region around the possible mutation and subject it to a restriction enzyme.

If the predicted fragment occurs, the mutation is present which can be a diagnostic tool

45
Q

Applications for Southern Blotting

Variable Number Tandem Repeat Analysis

A

Many areas of the genome vary in number from person to person. There are thousands of these regions that vary in number that is unique to each individual.

Tells you IF sequence is present and how much

DNA sequencing tells you what the sequence is.

46
Q

Applications in Medicine of RFLP and VNTR include

A

Looking for mutations

Embryo typing and genetic counseling for Huntingtons disease

Invitro fertilization

Remission test in cancer patients

Virus Detection

47
Q

qPCR

What is a key feature that helps qPCR work?

A

Measures the amount of PCR product after each reaction

A third primer is used that is complementary to sequences being amplified by the two primers. It contained flourophore and is bound by a quencher when that sequence is not present.

When wanted PCR is product is generated, the third probe can attach it self to the PCR product as the third primer should be complementary to the product. This releases the quencher and allows flourophor to activate.

48
Q

qRT-PCR

A

Measures RNA levels of specific transcripts (expression of RNA) giving idea of expression changes in disease or experimental conditions.

49
Q

Western Blotting

A

Western Blotting separates proteins by weight on a gel. It is transferred to a nitrocellulose membrane and incubated with an antibody that recognizes a specific proteins.

Various assays can be used to detect the antibody and its levels.

Not used for medical or diagnostic purposes!

50
Q

Immunohistochemistry

A

Asses protein abundance in cell or tissue including diseased tissue.

Requires LITTLE product

Used for medical and diagnostic purposes

51
Q

SNP

A

Single nucleotide polymorphisms

They cause phenotypic differences between people including resistance to human disease or sensitivity to a disease.

SNPs can be used to map disease loci if compared across several samples of the same disease

Genomic DNA is cut with restriction enzyme and amplified using one pair of PCR primers after being ligated into separate fragments. Products are labeled flourescently and hybridized to a microarray with oligonucleotides of each polymorphism to tag them.

52
Q

RNA-Seq

A

Utilizes SNP process but at a TRANSCRIBED level of SNP.

Can be used to identify heterogeneous cell populations.

53
Q

Mass Spectometry

A

Determine relative protein expression profiles of proteins in and out of cells as well as differences in protein modification.

Uses AA sequence!

Requires high quantity of material.

Biochemistry (18 decks)

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