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Biochemistry > DNA to RNA to Protein > Flashcards

DNA to RNA to Protein Flashcards

1
Q

Nucleotide Structure

A
  • The primary role of nucleotides is to preserve and transmit genetic information in living cells.
  • They also have other rules that include energy storage and transmission, as well as signalling.
  • In some cases they can even act as antioxidants
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2
Q

Three Parts of a Nucleotide (Sugar)

A
  • Sugar
    • Either ribose, or deoxyribose
    • The difference between these sugars is whats attached to carbon 2
    • Ribose has a hydroxyl group (-OH)
    • Deoxyribose only has a Hydrogen atom
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3
Q

Three Parts of a nucleotide (Nitrogenous Base)

A
  • Nitrogenous bases contain multiple nitrogen atoms in their aromatic rings
  • The nitrogenous bases are attached to the sugar at carbon 1 in the the ring
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4
Q

Three parts of a Nucleotide (Phosphate Groups)

A
  • The nucleotide has one, two or three phosphate groups attached to carbon 5 of the ring.
  • This is the difference between a NUCLEOTIDE and a NUCLEOSIDE
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5
Q

Nucleoside

A
  • Has a sugar and a base but no phosphate group
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6
Q

Covalent Bonds and Nucleotides

A
  • Nucleotides can form covalent bonds to other nucleotides, to make a chain.
  • Nucleotides with ribose sugars are called ribonucleic acids, and can be attached to other ribose sugars to make RNA.
  • Cells use RNA to make proteins
  • Deoxyribonucleic acids made from nucleotides with deoxyribose form DNA, which makes up the genome found in all living cells.
  • We say RNA and DNA have suagr-phosphate backbones because the covalent bonds that connect nucleotides, join the phosphate of one nucleotide, to the sugar of another
  • The nitrogenous bases that are attached to the ribose and deoxyribose sugars are often called residues.
    • They are categorized into two groups:
      • Pyrimidines
      • Purines
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7
Q

Purine Resides

A
  • Adenine (A)
  • Guanine (G)
    • Each contain two rings
    • 6-membered ring fused to a 5-membered ring
  • Remember them by thinking purine is the shorter name but the longer molecule
  • “Pure as Gold”
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8
Q

Pyrimidines

A
  • Cytosine (C)
  • Uracil (U)
  • Thymine (T)
    • Each have just one 6-membered ring
  • Remember them by thinking pyrimidine is the longer name but the shorter molecule
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9
Q

Deoxyribonucleic Acid (DNA)

A
  • Have a hydrogen, rather than OH group on carbon 2 of the sugar, and either pyrimidine or purine attached to carbon 1
  • There are phosphates attached to carbon 5, that one thats not in the ring
  • To create DNA, nucleotides are hooked together with a phosphodiester bond
    • They each lose two of the phosphates in the process
    • Note that the phosphate attached to carbon 3 of one nucleotide attached to carbon 5 of the next nucleotide
    • This is the basic of the sugar-phosphate backbone of DNA.
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10
Q

The Double Helix

A
  • Two DNA strands can join together to make one double stranded DNA molecule
  • Easton and Crick described the DNA as a helix → A double Helix
  • These two strand twist into a helix because of how the various parts of DNA interact with water.
    • The nitrogenous bases are hydrophobic aromatic rings (they want to be away from the water)
    • While the charged phosphates are hydrophilic and very happy to be in contact with water
  • DNA also has two grooves, a major groove and a minor groove, due to how the base pairs are orientated across the helix
  • The atoms exposed in the grooves are accessible to solvents and to interactions with proteins, and even some medicines.
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11
Q

Base Pairing Specificity

A
  • When DNA strands come together to make a double helix, there need to be interactions between the strands to hold them together.
    • Here those intermolecular interactions are hydrogen bonds between the nitrogenous bases.
  • The formation of these bonds is usually called base pairing
  • In order for string base pairing, the sequences must be complementary
    • In a complementary strand, a purine always pairs with the pyrimidine
  • In RNA, thymine is replaced by urical, which give us the mnemonic “CUT the Py”
  • Adenine and thymine are paired with two hydrogen bonds
  • Guanine and cytosine are paired with three hydrogen bonds
    • C and G are always together
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12
Q

DNA denaturation

A
  • The process of separating the two strand of DNA into single stands
  • This is also often described as “DNA melting”
  • Cells of enzymes called helicase that separate the DNA strand
    • It breaks the hydrogen bonds that hold the two strands together
  • Another way to separate the strands is by using heat
    • To make sure that the hydrogen bonds between the bases are broken, DNA is usually heated to around 100 C to make sure that all teh hydrogen bonds between G and C residue are broken
    • The bonds between A and T break at significantly lower temperatures
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13
Q

DNA Reannealing

A
  • The process of two single DNA strands that have been separated by helicase or heat coming back together to form the original double stranded DNA.
  • This is favoured as it keeps the hydrophobic bases away from water
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14
Q

DNA Hybridization

A
  • When a complementary (sometimes shorter) strand of DNA is annealed to another of interest
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14
Q

DNA Hybridization

A
  • When a complementary (sometimes shorter) strand of DNA is annealed to another of interest
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15
Q

Central Dogma

A
  • Explains the flow of information in all biological system
  • DNA makes RNA through transcription
  • RNA makes proteins through subsequents fancy process called translation
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16
Q

Genetic Code

A
  • Is the set of rules used by cells to translate mRNA messages into strings of amino acids in proteins.
  • One of those rules is that it tales 3 bases, or nucleotides to specify each amino acid.
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17
Q

Codon-Anticodon Relationship

A
  • Codons are found in mRNA that was transcribed from DNA, but anticodons are found on transfer RNA or tRNA molecules
  • During the process of translation, each codon is recognized by a complementary tRNA anticodon, with is specified amino acid attached to that same tRNA molecule.
  • Since we have four different bases, G, C, A and U there are 64 possible codons
  • Each of those 64 codons is specific and unambiguous, corresponding to precisely one amino acid
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18
Q

Degenerate Code

A
  • When the genetic code has more than one codon that can correspond to the same amino acid, thats how there are 64 codons specifying only 20 amino acids
  • Each codon still just specifies for one amino acid, and that is why the genetic code is unambiguous
  • This degeneracy relates to another feature of the genetic code called wobble
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19
Q

Wobble Pairing

A
  • When multiple codons code for the same amino acid, most often those codons share the same first two bases.
  • Those first two bases are thus the most significant in specifying the correct amino acid as originally coded.
  • The third nucleotide of the codon is the variable one, and is generally called the wobble position
    • This is due to the tRNA anticodon literally wobbling in matching the final nucleotide to the mRNA codon.
    • This setup is an evolutionary development that protects us against mutations.
    • If a mutation occurs in the wobble position, or a slightly wrong tRNA is brought in, that change os likely to be a silent mutation, meaning it has no effect on the polypeptide sequence
    • The first two bases will usually indicate the correct amino acid, and the protein will be produced normally
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20
Q

Point Mutations

A
  • A mutation affecting only one nucleotide in a gene sequence
  • Silent mutations altering a single nucleotide in the wobble position are an example of a point mutation that still made the same amino acid.
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21
Q

Missense, Nonsense Codons

A
  • Point mutations that can have significant effects:
    • A missence mutation is a mutation where one amino acid is substituted for another
      • Since they alter the primary amino acid sequence of the protein, we call them expressed mutations
      • These don’t always have to adverse, but it’s rare for missense mutations be be beneficial
    • Nonsense Mutation are even more serious than missense mutation, because they change a codon from an amino acid, to code for a premature stop codon instead
      • They are also known as truncation mutations, because they prematurely truncate or end the amino acid sequence
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22
Q

Initiation, Termination Codons

A
  • Start codons
    • AUG (School starts in August)
      • Signals where to start translation of mRNA to protein
      • Codes for methionine, which means that every preprocessed eukaryotic protein begins with methionine
  • Three Stop Codons
    • UGA (U Go Away)
    • UAA ( U Are Annoying)
    • UAG (U Are Gone)
    • The stop codons signal that protein translation should be terminated
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23
Q

mRNA

A
  • Messanger RNA
  • Carries the message encoded in the DNA to the ribosomes where that message can be translated into proteins
  • This process of DNA to mRNA is transcription
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24
Q

Three Stages of Transcription

A
  1. Initiation
  2. Elongation
  3. Termination
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25
Q

Initiation

A
  • The start of transcription
  • Here, RNA polymerase binds onto the DNA many base pairs in advance of the actual start site of transcription
  • This upstream area of DNA is called the promoter region of the gene
  • Proteins called transcription factors may bind to this region to help signal that RNA polymerase should bind as well
  • It is important that RNA polymerase knows exactly where to bind
  • Most eukaryotic and prokaryotic genes have a sequence of bases that is rich in T and N nucleotides that RNA polymerase recognizes and binds onto
  • In eukaryotes, this region that is rich in T and A nucleotides is often called the TATA box and is located 25 to 35 base pairs upstream of the actual start site of transcription
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26
Q

TATA Box

A
  • “ ta ta” → goodbye
  • RAN polymerase is saying ta ta to the promoter region as the polymerase moves on to start transcription
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27
Q

Pribnow Box

A
  • Essentially the same as the TATA box in eukaryotic cells, but is it is technically called the Pribnow box, or sometimes called minus 10 sequence, since in prokaryotes this sequence is 10 base pairs upstream of the start site.
  • The point is that both prokaryotes and eukaryotes have TA rich sequence somewhat upstream from the start site, and thats how RNA polymerase knows where to bind and get its running start.
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28
Q

RNA polymerase reading the DNA

A
  • DNA is a double helix that is connected by hydrogen bonds
  • Thus the DNA has to temporarily pulled apart so that RNA polymerase can read the DNA and make the polymer of RNA.
  • This unwinding is accomplished by helicase, which is a subunit of RNA polymerase itself
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29
Q

Unwinding of DNA

A
  • The unwinding happens when RNA polymerase binds onto the promotor region; this helps so that everything is ready to go by the same time the polymerase starts to leave the promoter region and head to the actual gene.
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30
Q

Transcription

A
  • RNA polymerase lurches forward, and when it hits the first base to be transcribed, RNA polymerase reads that base and matches it with the complementary base, which sticks right onto the DNA
  • So if the first DNA read is C, the first RNA base matches will be G, the complement.
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31
Q

Elongation

A
  • In this stage, the RNA strand is grown, i.e, elongated
  • RNA polymerase keeps reading the DNA and matching the correct RNA base pair, while simultaneously catalyzing a new phosphodiester backbone between the growing RNA strand and the next RNA nucleotide.
32
Q

Sense Strand of DNA

A
  • If our final RNA product has an AUG, for example then one of the DNA strands must have ATG
  • This means that the final RNA product “looks like” one of the strands of DNA.
    • We call that strand the sense strand because it has the same sequence as the RNA product, so in that way you can read it and it makes sense
  • In short, sense stand carries the code for the gene
33
Q

Antisense Strand of DNA

A
  • The RNA polymerase does not actually read and transcribe the sense stand
    • If the RNA product reads AUG, and the sense strand reads ATG, well RNA polymerase needs to read the other strand, which carries the complement of ATG.
    • It needs to read the strand that has TAC, the complement
    • So the polymerase reads TAC, and produces the desires AUG.
    • We call this complementary DNA strand, the antisense strand, not because it does not make sense, but because it is the exact opposite, the exact complement.
    • The antisense strand is also sometimes called the template strand
      • This name tells you exactly what this strand is: it is the template for transcription.
  • In short template strand actually gets read and transcribed
34
Q

Termination

A
  • Occurs though one of several mechanisms, and the exact mechanism depends on the exact organism and gene being transcribed
  • Prokaryotes, begin similar organisms, use one of two mechanisms for termination
    • Rho-Dependent
    • Rho-Independent
35
Q

Rho- dependent termination (in prokaryotes)

A
  • Rho is protein that combines onto a specific sequence of RNA and the binding of the rho protein introduces stearic strain which tugs on the RNA and pulls it away from the RNA polymerase.
  • In a prokaryotes, the RNA is now immediately ready to be translated
  • Meanwhile, the RNA polymerase finishes the job by sealing together the DNA strands and detaching to go find another gene to transcribe. This is the mechanism called Rho-dependent termination
36
Q

Rho-Independent Termination (in prokaryotes)

A
  • Does not use one of these proteins
  • Instead, baked into the DNA itself is a GC rich sequence, which turns into a GC rich sequence of mRNA
  • Since G and C are complements, this sequence is designed to fold onto itself introducing a little loop in the RNA
  • This little loops causes steric strain, tugging the RNA away from the DNA
  • Major takeaway:
    • termination can occur using proteins or can be protein independent, but in either case it requires strain on the RNA that tugs the RNA away from the polymerase
37
Q

Eukaryotic Transcription

A
  • Many mechanisms
  • All mechanisms use some kind of strain to pull RNA away
  • Most mechanisms require proteins to introduce that strain and trigger termination
38
Q

Post-Transcriptional Processing

A
  • Eukaryotes have a nucleus, which has one advantage that they can further process RNA before its sent out of the nucleus and into the cytoplasm for translation. (Process = Post-transcriptional processing)
  • Three major modifications
    • 5 prime modification
    • 3 prime modification
    • The removal of introns
39
Q

5’ modification

A
  • Is the addition of a methylguanosine cap
  • This cap is literally a guanine nucleotide attached backwards
  • The purpose of this cap is to prevent degradation of the mRNA transcript
  • See, viruses attack cells by inserting genetic material
    • So, once a transcript even from the cell has been utilized, we want it removed
  • For these reason out sells keep exonuclease around in the cytoplasm to chew up and degrade any nucleic messages
  • These enzymes work just like packman, they start at one end of the RNA polymer and break off each nucleic acid in turn
    • By simply adding a backwards guanine nucleotide (5 prime cap) the terminal nucleotide now does not fit into the active site of these exonuclease and this protects the mRNA from degradation
40
Q

3’ modification

A
  • The 3’ Poly A tail
    • Here a very long sequence of adenine residues is added to the 3 prime end of the mRNA.
  • This is also done to prevent degradation, but in this case, the exonuclease can start chewing off these adenine residues one by one
  • but now it takes significant time for the exonuclease to reach the singling part of the mRNA which means the exonuclease are occupied with the poly-A tail, and that gives time for the singling part of mRNA to be translated
  • This way the mRNA molecule does spend some time in the cytoplasm getting translated, but it doesn’t hang around forever.
  • In fact a cell can modify the length of the poly-A tail to produce more or less protein
    • A longer poly-A tail means the message will stay around longer, so it can be translated many times, resulting in more total protein
    • A short poly-A tail means the message will last less time
41
Q

Intron Removal

A
  • These introns are sequences scattered in between the exons, which are actual coding sequences
  • Exons EXIT the nucleus and are EXPRESSED
  • INTRONS are removed, so introns stay in the nucleus
  • The process of removing and joining the exons together is called splicing
    • Evidence that it suggests that a single gene might be spliced in different ways resulting in different proteins
    • So a given so called intron in one splicing might actually be an exon in a different splicing.
    • This extra complexity give eukaryotes greater access to a larger variety of proteins
42
Q

Translation

A
  • Process to create proteins
  • There are several different RNAs that work together to make protiens.
    • mRNA
    • tRNA
    • rRNA
43
Q

mRNA

A
  • Messenger RNA, and is is what was created during the transcription process
    • carries a message that will be decoded to create proteins
    • Each codon is decoded into one amino acid
    • has the same bases as DNA, except in place of thymine, it uses uracil
44
Q

tRNA

A
  • Transfer RNA
  • Responsible for bringing the right amino acid to the corresponding mRNA codon.
  • The portion of the tRNA that bonds to the mRNA is the anti codon
    • The anti codon sequence is directly able to be paired with the mRNA
  • Each tRNA molecule carries an amino acid
  • The first amino acid for eukaryotes is methionine (Met), and N-formylmethionine (f-Met) in prokaryotes
  • To remember that this is the first amino acid, you can think when you’ve met someone, the first time you see some you MET them (Start amino acid)
  • Both of these are coded for by the sequence: AUG
  • the mRNA transcript moves alone, three different tRNA occupied different sites in the ribosomes subunit
    • The A site
    • The P site
    • the E site
45
Q

The A site

A
  • is where a new tRNA enters, carrying an amino acid
  • A, first letter of the alphabet, so the A site is the first site of entry for tRNA.
46
Q

The P site

A

Where the amino acid is added to the growing peptide chain.

Think peptide when you think of P

47
Q

The E site

A

Think exit when you think of the E site

This is where the last tRNA dissociates from the ribosome.

48
Q

rRNA

A
  • Stands for ribosomal RNA
  • rRNAS are a structural RNA that composed the large and small subunits of the ribosomes themselves.
  • THEY DO NOT CODE
49
Q

Basic Role and Structure of ribosomes

A
  • Basically protein factors
  • Responsible for building the peptide chain and creating the primary structure of a protein.
  • Primarily composed of different types of rRNAs, as well as other protein matter
  • They have two subunits:
    • Large subunit
      • The one that contains the A, P and E sites
    • Small subunit
      • Binds first
50
Q

How the subunits carry out the process of Protein synthesis

A
  • Initiation
  • Elongation
  • Termination
51
Q

Intiation

A
  • Start of translation
  • During initiation, the small subunit binds to the mRNA before the start codon
  • In prokaryotes, the small subunit binds to the Shine-Dalgarno sequence
  • In eukaryotes the small subunit binds to the five prime cap from the process of post-transcriptional modification
  • This binding signals the start of translation
  • The actual translation process doesn’t start until the paring of AUG on the mRNA strand and the respective anticodon with met (starting amino acids) bind together
  • Once this occurs, the large subunit locks into place, bringing the A, P and E sites with it
  • Then the strands start being read across
52
Q

Elongation

A
  • Begins after the strand is being read across
  • It is the process of elongating the peptide chain that is being carried at the P site.
  • An amino acid bound to tRNA, or an aminoacytl tRNA binds at the A site and is moved over to the P site, via an enzyme called peptidyltransferase
    • Think of peptide tranfer to help you remember the name and function
  • These steps repeat until the mRNA strans is done being read, which then starts termination
53
Q

Termination

A
  • Terminating to ending of translation
  • It is up to a set of three codons called stop codons,
  • These codons are:
    • UAA
    • UGA
  • *UAG**
  • When any ones of these three stop codons is read by the ribosome, it gives the clear signal that translation should stop
54
Q

Post-Translational Modification of Proteins

A
  • Now that the polypeptide chain is completed it is simply just floating around in the cytoplasm
  • It still needs to be modified and packaged up to be sent to different places
    • This is done by the help of the Chaperone proteins
55
Q

Chaperone proteins

A
  • Come alone to help the polypeptide
  • The proteins condensed the polypeptide into a 3D shape so that instead of just a floating string of amino acids, it becomes an actual recognizable protein
56
Q

Chromatin Structure

A
  • Each human cell has 46 chromosomes
  • To prevent chromosome entanglement, DNA molecules are wounded around a group of proteins known as histones, forming chromatin.
  • Around 200 base pairs of DNA are wound around one histone complex forming a nucleosome.
  • In this state the DNA molecule literally looks like beads on a string.
    • This form of chromatin is known as euchromatin, and allows the DNA molecule to be fairly accessed
    • This is crucial to allow RNA polymerase to bind to DNA and initiate transcription
  • Nucleosomes themselves can be further packages to form increasingly more compact chromatin
    • This compact form of chromatin is known as heterochromatin
  • Given how compact it it, genes and heterochromatin are pretty inaccessible to the proteins necessary for transcription
    • This gives cells a great tool to permanently shut off genes that are not necessary for its function.
57
Q

How the cell alters its Structure

A
  • One way is to alter the DNA molecule itself
  • Cytosine and adenine bases in DNA can be methylated
    • DNA methylation hinders the ability of RNA polymerase to transcribe and is also associated with more compacted DNA.
  • We can also modify our histone proteins to alter their ability to bind DNA
    • addition of methyl, acetyl or phosphate groups to histones decrease their affinity for DNA and makes the DNA molecule more accessible
    • Conversely, removing these groups makes the chromatin more compact, so its less available for transcription
    • By adding or removing these modification these cells can control which regions are accessible
    • The histones can also simply be displaced almost like sliding two beads apart, allowing the DNA sequence between them to be accessed more easily.
    • But accessible DNA alone isn’t enough to ensure transcription
58
Q

Transcription Factors and Transcriptional Regualtion

A
  • Transcription is catalyzed by RNA polymerase II, which binds DNA and creates a complementary RNA transcript.
  • But RNA polymerase on its own doesn’t know which gene to bind to and transcribe
    • It requires a little help from other proteins known as transcription factors
  • These factors bind to specific DNA sequences, known as response elements, which are located in the promoter regions of every gene.
  • There are two kinds of transcription factors:
    • General
    • Selective
59
Q

General Transcription Factors

A
  • Serve crucial roles in the transcription machinery, such as recruiting RNA polymerase and unwinding the DNA helix
  • Without these general factors, transcription could not work
60
Q

Selective Transcription Factors

A
  • Bind to specific response elements that are found in only some genes
  • These response elements are often found hundreds of bases away from the promoters of the gene and are known as enhancers.
  • Selective transcription factors give the cell an excellent way to regulate which genes are expressed.
  • In the presence of specific signals, these transcription factors bind to their response elements in the enhancer region and either promote or inhibit the transcription of those genes.
  • some transcription factors even, regular other transcription factors, so this regulation can be complex
61
Q

Post-Transcriptional Regulation

A
  • Freshly transcribed RNA contains exons, which hold the coding sequences for proteins, and introns, which are recognized and removed via slicing, leaving only exons in the final mRNA transcript
  • Some genes can be spliced in numerous different ways, giving us slightly different versions of the same protein.
  • Recall that the mRNA must be exported from the nucleus to the cytoplasm for translation.
    • No matter how many transcripts we have, if they are stuck in the nucleus, proteins won’t be made
  • The nucleus therefore can regulate which transcripts and how many are exported from the nucleus.
  • Once in the cytosplam, the transcript must also race against proteins which degrade RNA
  • The poly-A tail added to mature transcript helps to protect the mRNA from degradation
  • mRNA also contain untranslated regions or UTRs, on both the 5’ and 3’ ends of the transcripts.
    • While these regions are generally not translated into protein, they serve critical functions in regulating translation
    • For example, the ribosome relies on the 5’ UTR to bond to the mRNA transcript and intiate translation
    • The 3’ UTR often forms secondary structures that help stabilize the transcript or are recognized by specific enzymes that degrade the transcript.
    • When transcripts are quickly marked for degradation, less protein will actually get made.
    • All these factors work together to give the cell a very sophisticated gene regulation machinery
62
Q

Cancer

A
  • Many human cancers can be traced back to changes in gene expression pattern in cells
  • This is especially true of genes that regulate the cell cycle and cell division
  • Some genes known as oncogenes, promote cell division
    • Normally, these genes are suppressed in non dividing cells, preventing tumor formation
  • Many cancers arise from mutations that lead to overexpression of these oncogenes
63
Q

Tumor Suppressor genes

A
  • normally suppressed cell division, or are involved in DNA repair
  • When these genes are incorrectly turned off, cells either divide more frequently increasing the likelihood of tumor formation or accumulate DNA damage, making them more susceptible to tumor-promoting mutations
  • When these tumours cells acquire the ability to invade or migrate to the rest of the body, cancer develops
64
Q

Recombinant DNA

A
  • Is a DNA sequence created by combining genes from more than one source that would not normally occur in nature
    • A common way we do this is by combining a human gene with a bacterial plasmid, which is a circular, double stranded DNA that is genetically isolated from different species
65
Q

Carotenoid gene

A
  • For our carotenoid gene, we need the exact sequence of nucleotides that encodes for the gene
  • We can then artificially generate or isolate it from another plasmid that we know already contains the sequence using restriction enzymes.
66
Q

Transformation

A

Introducing new genetic information into a host so that the host can integrate the new DNA into its genome

67
Q

Restriction Enzymes

A
  • These enzymes are site specific, meaning that we can choose certain enzymes that will only latch onto a particular sequence of DNA on our plasmid
  • They can then cleave our double-stranded DNA, usually in an asymmetrical pattern, leaving behind two exposed sticky ends that can later hybridize with a new plasmid sequence
  • The restriction enzymes break up the circular plasmid into DNA fragments
68
Q

Polymerase Chain Reaction (PCR)

A
  • Once the plasma has been broken the PCR is used to determine which fragment is the carotenoid gene and how we can be able to create more.
  • It will use a set of primer, complementary DNA sequences that we design, to locate and bind to short sequences of DNA on out carotenoid genes, and then go through the process of DNA replication.
  • During PCR, the DNA strands go though cycles of denaturing, hybridization, and replication to exponentially create more of the carotenoid gene.
  • DNA replication is always happening in some solution, in both in out bodies and in a test tube.
69
Q

dNTP

A

the solution that DNA fragments are placed in and are the building blocks for more carotenoid sequences,

70
Q

Denaturing

A
  • Uses incredibly high temperatures to heat up our solution, and separate double-stranded DNA sequences into two parental single-stranded sequences
    • Typically heated to about 94 to 96 degrees c
71
Q

Hybridization

A
  • Solution is cooled
  • In this case the DNA strands will anneal to the primers we added, forming a short double stranded DNA hybrid.
  • This allows for polymerase to come in and use dNTPs to extend the sequence, effectively replicating the DNA in out replication step.
72
Q

Thermocycler

A
  • Machine where this whole process can take place
  • Regulates the reaction temperature to allow for the DNA to denature and then hybridize
  • To make sure that our polyermase doesn’t denature we use a special Taq polyermase, isolated from bacteria that live in incredibly high temperatures
    • The mixture has to be heated to at least 70 degress C so that the Taq polymerase to function optimally
73
Q

After a cycle

A
  • After each cycle, the carotenoid sequence will form a new duel stranded hybrid that can then get broken apart to be used as a template for another cycle, making more copies of our gene
  • If we look at the math, for every n cycles we produce 2 to the nth power stands of DNA
  • This means that after 30 cycles of PCR we would have created over a billion strands of carotenoid genes to work with.
74
Q

Gel Electrophoresis

A
  • Technique that can help us figure out how many base pairs long a segment of DNA is
  • Works by moving fragments of DNA though an agarose gel plate
  • The gel acts like a mesh barrier, so smaller pieces of DNA can move faster than larger ones
  • The DNA fragments all move forward because of the negatively charged phosphdiester backbone
  • All the DNA segments being tested at point A will be tested at point B because of a current running through the agarose gel
  • The negatively charged phospodiester backbone of each DNA fragment will move towards the positive end of the gel
    • The larger pieces will stay back and get stuck in the gel
    • The farther the DNA travels the smaller the size
75
Q

Southern Blotting

A
  • Use probes to identify the target DNA after it has been run on a gel. Useful for identifying if the target gene is in the sample
76
Q

Subcloning

A
  • A form of cloning that is the process of inserting genetic material into a plasmid, to make recombinant DNA
  • We want to make sure that we use a plasmid that will introduce or “knock-in” the gene.
  • One that will work here is the pAcc-B plasmid
    • In order to get it in, we first need to prep it for insertion with a restriction enzyme
77
Q

Restriction enzyme

A
  • Are site- specific, so enzymes will only latch onto a particular sequence of DNA on out plasmid
  • Here we would just use the same restriction enzyme that initially cut the sequence out of the original plasmid that held it since the sticky ends created would be the same
    • That way the sequences could line up and form our completed circle of recombinant DNA
  • To make sure that we’ve selected only the plasmid that contain our new carotenoid interest, we can take advantage of antibiotic restriction genes that are typically added into the plasmid sequence template
  • We do so by transferring out product to a bacteria like E.coli and then allow it to grow on antibiotic treated Agar plates, and only bacteria that have fully circularized plasmid containing the catenoid insert should grow.
    • We can then select these bacteria, allow the to replicate and extract enough of our carotenoid containing plasmid from them and repeat the transformation process to get it to integrate into our rice genome
  • Bacteria with plasmid grow and multiply.
  • Bacteria without plasmid fail to grow,
78
Q

Deamination of Nitrogenous Bases

A
  • Deamination is the replacement of an amine group with a carboxyl group.
  • This can either be spontaneous or enzyme-catalyzed, and these substitutions can have powerful effects because they change the hydrogen bond characteristics of the base.
  • They result in mutations: for example, deamination of the pyrimidine changes it to uracil.

Biochemistry (4 decks)

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