Molecular genetics Flashcards
Describe DNA structure:
- DNA=deoxyribonucleic acid
- Sugar and phosphate backbone
- Bases are attached to the sugars (changes in these bases lead to different genes)
- One set of sugar, base and phosphate is a nucleotide
- Two strands combine in a double helix to form DNA
Describe the structure of a nucleotide:
- Each nucleotide is composed of a 5-carbon sugar (deoxyribose), a phosphate group and a nitrogen-containing base
- The four bases possible in DNA are adenine (A), guanine (G), cytosine (C) and thymine (T)
- One strand of DNA is made of a chain of these nucleotides
Double Helix - history
- 1940s - Chargoff found that A and T and G and C are found in equal amounts in DNA
- Franklin’s experiments with X-rays and water showed that bases were on the inside and sugars and phosphates were on the outside
- 1953- Watson and Crick published paper describing double helix
Describe a double helix structure:
- Looks like a spiral shaped ladder
- The “rungs” are pairs of bases – held together by hydrogen bonds:
- Complimentary pairs – A always pairs with T and G always pairs with C
- Results in each strand being a compliment of the other – “antiparallel”
- The sides of the ladder are phosphates and sugars that are covalently bonded
- There is a 5’ and 3’ end to each strand of DNA (since strands are antiparallel there is one of each at each end
- (The bond between a base and a sugar is also covalent)
Describe RNA – ribonucleic acid:
- Single-stranded
- Pentose sugar is ribose instead of deoxyribose
- Uracil (U) replaces Thymine (T)
- Can form different structures – different types of RNA
- ie. mRNA, tRNA, rRNA
Describe genes and genomes:
- A heritable factor that consists of a length of DNA and influences a specific characteristic
- Structural Gene – section of a DNA strand that encodes for the production of one or more proteins and occupies a specific position on a chromosome (eukaryote) or circular DNA (prokaryote)
- Genome – sum of all DNA that is carried
- Human Genome Project – entire base sequence of human genes
- Chromosomes vary in size and number of genes but are generally several million bases long
Describe alleles:
- Various specific forms of a gene
- Differ from each other by one or only a few bases
- New alleles are formed by mutations
Describe Cairn’s Technique for determining length of DNA:
- Cells are grown in radioactive thymine (T nucleotide)
- Cells are lysed to isolate chromosomes
- Chromosomes are fixed to a photographic surface
- rinsed with radioactive-sensitve AgBr emulsion (silver ions stick to radioactive T)
- result appears black when film is developed – can visualize length of DNA as a result
Where Did nucleic acids get their name?
- Weak acidic material isolated from the nucleus
- First isolated from nuclei of white blood cells in 1869 (by Friedrich Miescher (but not studied thoroughly until about 100 yrs later)
- The two nucleic acids are DNA and RNA —> Deoxyribonucleic Acid and Ribonucleic Acid
Describe DNA: A Brief History
- 1928 – Griffith mouse studies
- 1944 – Avery, MacLeod, & McCarty –> DNA can turn harmless bacteria into killer bacteria
- 1952 – Hershey & Chase DNA is the genetic information
- 1952 – Rosalind Franklin X-ray diffraction shows helical structure of DNA (article)
- 1953 – Watson & Crick publish paper on complete double helix structure of DNA (using Franklin’s X-ray image!)
Describe Griffiths experiment - the transforming principle:
- 1928
- Study pathenogenic (pneumonia causing ) bacteria in mice
- Showed that something from killed pathenogenic bacteria must be transferred to live bacteria cells
- When heat-killed bacteria were treated with a DNA-digesting enzyme transformation did not occur
Describe Avery, MacLeod, and McCarty:
- 1944
- Avery and McCarty repeated and refined the experiments of Griffith to show that DNA and not Proteins were the hereditary material of bacteria.
Describe Hershey and Chase:
- 1952
- Experimented with transfer of DNA by a virus called a bacteriophage – infects bacteria
- Labeled protein coats and bacteria separately
- Infected bacteria with viruses then removed protein coats
- Bacteria showed evidence of viral DNA – was transferred
Describe Linus Pauling:
- Discovered the alpha helix (chain) in secondary structure of proteins and proposed that the DNA model is also a helix structure.
- 1953 proposed that DNA is made up of a triple helix structure.
- Rosalind Franklin, based on her work, realized that it cannot be a triple helix (and shared this information with Watson and Crick)
Describe Rosalind Franklin:
- 1920-1958
- X-ray diffraction - 1952
- Experimented with x-ray crystalography to study the structure of DNA
Describe Watson and Crick:
- With the help of one of Franklin’s images and a lot of collaboration were able to come up with double helix structure of DNA
- Also worked with models/model making
- Watson acquired the image in 1953 and Watson and Crick published their model of DNA in May of that year
- Won the nobel prize along with Wilkins (Franklin’s colleague that she did not get along with)
Describe Meselson and Stahl:
- 1957 – discovered that DNA replication is a semi conservative (more on this later) . By using the isotope nitrogen-15 on the original DNA strand of a bacteria culture, they then placed the culture in a test tube and let them grow by asexual reproduction.
- Nitrogen 15 glowed in the test tube.
- After a number of replication it was discovered that the glowing nitrogen 15 in the bacteria becomes dimmer and dimmer with each successive replication thus showing that only half of the original DNA strand (Nitrogen 15) was found in the new bacteria that were produced.
- Then they shifted bacteria to N-14 medium
- All new DNA was of intermediate density (combination of N-15 and N-14)
Describe DNA replication:
- In order for organisms to reproduce or grow, cells must divide —> DNA must be replicated
- Cells must have nucleotides available —> come from DNA in food you eat!
- DNA replication is semi-conservative —> new double helices each contain one original strand and one new strand
Define replicon:
a specific part of the DNA sequence where replication starts – may have many per strand
Define helicase:
enzymes that bind to DNA at replication origin and breaks the hydrogen bond between the two DNA strands and unravel segments of DNA (start at origin and move along the molecule
Define replication bubble:
Unwound open area
Define replication fork:
y- shaped area at each end of a replication bubble
Describe initiation:
- Replication
- Helicases
- Replication bubble
- Replication fork
- Single strands in a bubble are used as a template for nee DNA
Describe elongation:
- DNA polymerase (III) – enzyme that inserts into replication bubble to add nucleotides to the new strands, one at a time
- Nucleotides are added to complement those on the old strand (eg. A goes with T)
- Elongation – the process of creating the new strand of DNA
What are the rules for elongation?
- DNA polymerase (III) only works in the 5’ to 3’ direction
- Primer must serve as the starting point – short piece of RNA
Describe the leading vs. lagging strand:
- Because strands are complementary, 5’ and 3’ ends of each strand in a double-stranded DNA are opposite
- Elongation can only occur in the 5’ to 3’ direction
- If elongation needs to occur in the 3’ to 5’ direction on a strand it does this by short segments (run 5’ to 3’) that are spliced together later
- Leading strand – the one that is actually being synthesized in the 5’ to 3’ direction
- Lagging strand – the strand that needs to be synthesized using Okazaki fragments (because the main direction of replication for the molecule is in the ‘wrong’ direction for this strand
- Okazaki fragments are spliced together by DNA ligase
- Also many other protein enzymes involved
Describe the primer:
- Elongation needs to start at a primer (this is where DNA Polymerase must start from)
- Primer- short strand of RNA
- Complementary bases to the strand of DNA that encodes for it
- Necessary because nucleotides can only be added to other nucleotides
- Complementary bases to the strand of DNA that encodes for it
- Primase (a type of RNA polymerase) – the enzyme that creates the RNA primer
- Primer is removed by DNA polymerase (I) once elongation starts
Describe proofreading:
- After a new nucleotide is added, DNA polymerase (II) checks to ensure hydrogen bonding is taking place (if it isn’t than the bases aren’t correctly paired
- DNA polymerase II can excise the incorrect base and replace it with the correct one
Describe the DNA replication machine:
A number of enzymes are required to copy DNA…
- Helicase –> unzips (unwinds) double helix
- Primase –> synthesizes RNA primer to initiate replication site
- DNA Polymerases –> add new nucleotides, remove primer, and checks for mistakes
- Ligase –> splices Okazaki fragments together when synthesis complete
- Many other proteins and enzymes involved
Describe termination;
Completion of new strands and dismantling of replication machine
Describe PCR:
Polymerase Chain Reaction
- Purpose –> to create many copies of a fragment of DNA. (copying)
- Allows for multiple testing of the same strand of DNA
- Application in forensic studies
Describe the process of PCR:
- Heat DNA to Denature it (to have the DNA uncoil)
- Expose to a heat resistant polymerase (TAQ polymerase), primers and DNA nucleotides
- Polymerase copies the DNA
- Allow to cool (DNA re-anneals –> reforms)
- Repeat this cycle many times
Describe restriction enzymes:
- Recognize a specific sequence in DNA and cleave at that sequence
- For example, you may have a restriction enzyme that cleaves between the A and A in a TAAGC sequence
- Because these patterns will show up in different places in different DNA sequences (sequences are unique in an individual), will end up with different lengths of fragments
Describe gel electrophoresis:
- DNA is slightly negative
- Has been cut into different length fragments by restriction enzymes
- Wells are cut into the gel – DNA is placed in wells
- Electric current is applied to the gel to pull DNA pieces through
- DNA pulled to positive charge
Shorter segments move further along the gel
What do the bands on a gel mean?
- The ones that travel furthest are the shortest pieces
- The closer the restriction enzyme sites, the shorter the resulting fragment
- Can help you to determine a pattern of restriction enzyme sites in DNA
- Certain sequences (fragment lengths) will be passed on by one of the parents
What are the steps for DNA fingerprinting?
- Restriction enzymes are used to cut up a sample of your DNA
- The cut DNA is separated using electrophoresis – the longer the strand, the slower it will move
- The DNA is compared with other DNA – are you the father? If so, you will share some of the bands with your child (as does the mother), are you the culprit? If so, your bands will match the bands of the criminal’s DNA
Describe how PCR is connected to DNA fingerprinting:
(PCR – polymerase chain reaction) allows REALLY small samples of DNA to be used in fingerprinting – PCR will copy DNA outside of a cell as long as nucleotides and enzymes are present
What is a protein made of?
- Building blocks are amino acids
- String of amino acids makes the primary structure of Proteins
- Primary Structure folds into alpha helix or beta sheet (secondary structure)
- Tertiary structural proteins are formed by combinations of helixes or sheets from a single structural gene.
- Quaternary structural proteins are complicated proteins (fibrous or globular) that are made from more than one structural gene.
Describe the genetic code:
- Order of base pairs in a strand of DNA
- DNA includes genes and regions of non-coding DNA
- In genes - every group of three bases either codes for a specific amino acid or a stop codon
- Order of amino acids will dictate the function of the protein
- Order of amino acids makes up primary structure of protein
Describe gene expression:
- The process of making a protein from DNA
- Information is copied from DNA to mRNA (type of RNA) to protein
- Process of creating mRNA from DNA is called transcription
- Process of creating protein in ribosomes (free floating ribosomes in the cytoplasm or on RER) from mRNA is called translation
- Order of bases –> determines order of amino acids –> determines polypeptide –> determines protein!
- Group of three bases = codon
Describe the central dogma:
- DNA cannot leave nucleus, but protein synthesis occurs in cytoplasm - how can this be?
- There must be an intermediate: DNA –> mRNA –> protein transcription translation
What are the types of RNA?
- mRNA
- tRNA
- rRNA –
Describe nRNA:
Messenger RNA – intermediate RNA strand that forms the code for amino acids
Describe tRNA:
Transfer RNA – attached to amino acids to match specific amino acids up with their code
Describe rRNA:
Ribosomal RNA – major components of ribosomes
Describe genetic code:
- Genetic code is universal as all organisms including adenoviruses (viruses that contain DNA), use the same 4 letter nitrogenous bases (A,T, C, G) to sequence all genes)
- Genetic code is transcribed from DNA to mRNA sequence and is read in 3 letter sequences called codons
- More than one codon can encode for the same amino acid. This allows for the possibility of errors in the genetic code to not always result in a physical error to the organism.
Describe start codons:
Same as codon for methionine – all proteins will start with the amino acid methionine
Describe stop codons:
Three –do not code for proteins, only tell protein synthesis to stop
How do you read a codon chart?
Find 1st base, follow it to the column for the second base then find the codon that ends in the correct third bas
Describe sense vs anti-sense strands of DNA:
- Sense– the strand that contains the gene in question and is transcribed (used as a template to make mRNA)
- Anti-sense strand – the other strand (complement of the antisense strand)
Describe transcription:
- Process of making mRNA
- Information of DNA copied into mRNA (a complement of the antisense strand of DNA)
- mRNA then moves from nucleus into cytoplasm
- RNA polymerase (II) – main enzyme involved (a sequence on the DNA called the promoter tells RNA Polymerase II where to bind)
How does RNA polymerase work?
- Binds to promoter on antisense strand (Promoter sequence comes before the gene and tells the RNA polymerase where to start)
- Section of double helix opens
- RNA Polymerase (II) moves along DNA and completes a strand of mRNA complementary to DNA
- Copies the DNA in 5’ to 3’ direction
- Adds one nucleotide at a time (uracil (U) instead of thymine (T))
- Reaches a sequence that signals transcription to stop (terminator sequence)
- mRNA released and travels to cytoplasm
- DNA recoils
Describe translation:
- Codons on mRNA are translated into a sequence of amino acids
- mRNA must move from nucleus to ribosomes (free floating in cytoplasm or RER), before translation can occur
- tRNA is attached to each amino acid and matches it up with it’s code (anticodon)
Describe ribosomes:
- Small organelles made mainly of rRNA (and some proteins)
- Made of a large and small subunit
- Match tRNA with the corresponding mRNA
- Bring in enzymes to form bonds between amino acids
- Free Ribosomes synthesize proteins for use in the cell, those on RER synthesize proteins for excretion
What are the steps for translation?
- mRNA binds to ribosome so that two codons are exposed
- tRNA with methionine is first to bind
- Second tRNA (to match second codon) brings in second amino acid
- Enzymes catalyze peptide bond formation
- Ribosome moves one codon along chain and process repeats
- Continues until stop codon reached
- At stop codon, chain is released and ribosome complex disassembles
Define mutation:
Permanent change in genetic material of an organism
Describe somatic cell muations:
- Mutations in body cells
- Not passed to next generation - but can lead to cancer
Describe germ line mutations:
- Mutations in reproductive cells
- Passed from one generation to the next
Define gene:
A heritable factor that controls a specific characteristic
Define allele:
A specific form of a gene (differs from other alleles by only a few bases and occupies the same location on the same chromosome number)
Define genome:
Whole genetic information of an organism
What are the areas where mutations can occur?
- Germinal Mutation
- No affect on the carrier but offspring affected.
- Somatic Mutation
- Carrier affected but no affect on offspring
- Embryonic Mutation
- Can result in a germinal or somatic mutation
What are the types of genetic mutations?
- Chromosomal mutation
- Point mutation
Describe chromosomal genetic mutation:
Rearrangement of genes – usually by crossover or by loss or duplication of portions of chromosomes (ie Non Disjunction, Translocation, Inversion, etc…)
- Almost always lethal and found in many cancer cells.
Describe point genetic mutation:
Substitution of one nucleotide for another or insertion or deletion of nucleotides – leads to one of the following:
A. Substitutions: one nucleotide for another
B. Frameshift mutations
- Insertions (additions): an extra base is slipped in
- Deletions: a base is missing
Describe base substitutions point genetic mutations:
- Usually non lethal but can be lethal.
- Ie. Silent mutation (no overall effect), nonsense mutation (causes a premature stop codon) or missense mutation (change in an amino acid of a protein
Describe frameshift point genetic mutations:
Usually caused by addition or deletion of an amino acid –> shifts codons so entirely new amino acids are made. Potentially lethal depending on the location
Describe sickle cell anemia:
An example of a base substitution mutation to the hemoglobin molecule that is harmful
Describe a missense genetic mutation:
- Substitution mutation
- Results in an altered protein (eg. sickle cell anemia)
Describe a silent genetic mutation:
- Substitution mutation
- Has no effect on a cells metabolism
Describe a nonsense genetic mutation:
- Substitution mutation
- Renders gene unable to code for a functional polypeptide
Describe serious genetic mutations:
- Insertions or deletions are most serious – especially if frameshift occurs
- Non-sense can also be serious
- Mis-sense are usually not as serious and are usually caused by substitutions
- If a substitution changes a regulatory sequence (start or stop codon) or an amino acid essential to structure, will be much more serious
- Silent mutations are not generally a concern (are possible because of redundancy of code)
How can genetic mutations be beneficial?
- Mis-sense mutations especially can help organisms develop new proteins that can lead to favorable traits
- Leads to genetic variation
- Also plays a role in the variety of antibodies in immune system
What causes genetic mutations?
- Mutations may be spontaneous (DNA polymerase incorrectly pairing nucleotides) or induced by a mutagen
- Radiation– cause physical changes in structure of DNA
- Chemical mutagens - enters the cells and reacts chemically with DNA to cause mutations
- Infectious: bacterial or viral pathogen
Describe radiation:
- X-Rays, short wave or UV radiation
- High-energy rays tear ( ionizes )DNA molecule causing physical changes
- (UV light can also cause chemical reactions between C and T after it reacts to water to form hydrogen peroxide )
Describe chemical mutagens:
- Usually mimics a part of DNA and inserts itself chemically - usually different base-pairing properties
- Can cause incorrect nucleotide insertion during replication
- Most are carcinogens – cause cancer
- Examples: nitrites, gasoline fumes, chemicals in cigarette smoke
Describe genetic mutations in cancer:
- Mutations can accumulate
- Most cancers are caused by combinations of mutations
Describe mitochondrial DNA:
- Mitochondria have the same nitrogenous bases as genomic DNA but has different number of rRNA and tRNA than genomic DNA
- Passed on only from mother
- Can compare sequences to trace lineages
- Different families have different DNA due to mutations over time
Describe recombinant DNA:
- Molecule of DNA that includes genetic material from different sources
- Used to alter the genetics of an organism
- The basis behind genetic engineering
Describe restriction endonucleases:
- Restriction enzymes that cut somewhere in the DNA strand (rather than the ends)
- Cut DNA in the middle of a specific sequence of bases – target sequence
- Restriction site – where the nuclease cuts
Describe sticky ends:
- If a restriction endonuclease leaves staggered ends, we call them “sticky ends”
- Leave a few unpaired bases
- Better for recombination
Describe the process of genetic engineering:
- Restriction endonuclease is used to cut desired gene out of donor DNA
- Same restriction endonuclease is used to remove a portion of the recipient DNA
3.The single base pairs of the sticky ends match up – one segment of DNA fits into the other like a puzzle piece - Ligase is used to join the pieces of DNA together
