DNA and BioTechnology Flashcards
Nucleotide
Consists of a nitrogenous base covalently attached to a sugar (ribose or deoxyribose) and one to three phosphate groups.
Nucleoside
Consists of a nitrogenous base covalently attached to a sugar (ribose or deoxyribose) but without the phosphate group
DNA
- Deoxyribose sugar
- Double stranded
- Uses A, T, C, G
**When replicating DNA cannot complete the synthesis of 5’ end so the chromosomes will shorten
RNA
- Ribose sugar
- Single Stranded
- Uses A, U, C, G
DNA Backbone
Phosphate linked to 3’C of one sugar and 5’ phosphate group of the incoming sugar
Direction to Read DNA
5’ —> 3’
5’ will have a Free OH or Phosphate on C5
3’ will have Free OH on C3
Aromaticity
Stable ring structures that are unreactive
- Cyclic
- Planar
- Conjugated
- Follow Huckel’s Rule (4n+2 pi electrons)
- Generally form a type of S and P hybridization orbitals causing delocalizations of the electrons adding to stability
Purines
Contain two rings
- Adenine (A)
- Guanine (G)
Pyrimidines
Contains 1 ring
- Cytosine (C)
- Thymine (T)
- Uracil (U)
Watson Crick Model
- Double Helix: 2 strands of DNA that run antiparallel
- Sugar phosphate (alternating) backbone on the outside and nitrogenous bases inside
- Complementary base pairing A-T (2 H bonds) and C-G (3 H bonds)
- DNA is ALWAYS read 5’ –> 3’
Chargaffs Rule
Purines (A,G) = Pyrmidines (T, C) and complements match meaning %A = %T and %C = %G
***Does not hold for RNA
B-DNA
Right handed helix with major and minor grooves between interlocking strand being site of protein binding
Z-DNA
Left handed zig zag helix, high G, C content or [salt] is high
Denaturing
Separating 2 strands of DNA without altering other aspects (like its nucleotides, sugars, phosphates, etc…)
Reanneal
Denaturing conditions are slowly removed so that DNA comes together
Histone
Small basic protein that chromosomes wind around forming chromatin
- Proteins include H2A, H2B, H3, and H4
- H1 = not part of histone but seals DNA off
Nucleosome
DNA and the histone proteins in a compact and stable structure
Heterochromatin
Chromosome material of different density from normal in which the activity of the genes is modified or suppressed.
- Dense and Compacted chromatin
- Dark appearance in microscope
- Silent Transcriptional Activity
- DNA is highly repetitive
Euchromatin
Chromosome material that does not stain strongly except during cell division. It represents the major genes and is involved in transcription.
- Uncondensed and Dispersed chromatin
- Light appearance in microscope
- Expressed Transcriptional Activity
- Genetically Active DNA
Telomere
Repeating Unit (TTAGGG) cap at the end of each DNA strand that protects our chromosomes. High in GC content prevent unraveling
Telomerase
Enzyme that replaces the sequence lost in each replication. Highly expressed in rapidly dividing cells; Synthesizes telomeres
Centromeres
Region of DNA found in the center of chromosomes
Replisome
Specialized proteins that assist the DNA polymerases
Helicase
Unwinds DNA double helix
Primase
Synthesis of RNA primer to begin DNA replication (in the 5’ –> 3’ direction)
DNA Polymerase Alpha and Delta
Eukaryotes:
Adds nucleotide to the growing daughter strand; synthesis of DNA
DNA Polymerase III
Prokaryotes:
Adds nucleotide in the growing daughter strand; synthesis of DNA
DNA Topoisomerase II (DNA Gyrase)
Reduces torsional strain from + Supercoils by introducing nicks in the DNA
Removes + Supercoils ahead of advancing replication forks
RNase H
Eukaryotes:
Fills gaps left after RNA primer excision
5’ —> 3’ Exonuclease
DNA Polymerase I
Prokaryotes:
Fills gaps left after RNA primer excision
5’ —> 3’ Exonuclease
DNA Ligase
Joins DNA Okazaki fragments
Single Stranded DNA Binding Protein
Prevents Reannealing of DNA double helix and degradation by nucleases during replication; stabilizes the unwound template strands
Supercoiling
Wrapping of DNA on itself as the helical structure is pushed closer to the telomere
Semiconservative Replication
One parental strand is retained in each of the two resulting daughter strands
Prokaryote Replication
Leading Strand 5’ –> 3’
Lagging Strand 3’ –> 5’ by creating okazaki fragments
One origin of replication
Eukaryote Replication
Leading Strand 5’ –> 3’
Lagging Strand 3’ –> 5’ by creating okazaki fragments
Contains multiple origins of replication - allow many parts of the DNA to be replicated simultaneously
Cancer
Proliferate excessively and not subjected to cell control
Metastasis
Cancer migrates to distant tissues through blood and lymphatics
Oncogenes
Mutated genes that cause cancer
Proto-Oncogenes
Precursor to oncogenes, before they become mutated
Anti-Oncogenes
Tumor suppressor genes that inhibit the cell cycle or participate in gene repair processes
ProofReading Repair Mechanism
DNA Polymerase does the proofreading during the S phase of the cell cycle - incorrect base pairs are excised and replaced with the correct one
Mismatch Repair Mechanism
MSH2 / MCH1 do mismatch repair during the G2 cell cycle - detect and remove errors found in the S phase
Nucleotide Excision Repair Mechanism
Excision Endonuclease does nucleotide excision during the G1 and G2 phase of the cell cycle - Removing thymine dimers (caused by UV light) that distort the helix
Base Excision Repair Mechanism
Glycosylase and AP Endonuclease do Base Excision Repair during the G1 and G2 phase of the cell cycle - Remove uracil that was made by the cytosine deamination
Recombinant DNA
DNA fragment is composed of nucleotides from 2 different sources. Can be multiplied by cloning or PCR to study
Cloning
Introduces a fragment of DNA into a vector plasmid -
Produces large amounts of desired sequence
Restriction Enzymes (Endonuclease)
Recognize sequences and cut backbone of double helix
Sticky Ends
Hanging nucleotide - good for recombination of a restriction fragment with vector DNA; able to bind
DNA Libraries
Large collections of known sequence
Genomic DNA Library
Include coding (exon) and noncoding (intron) DNA
cDNA (Expression) Library
Reverse transcribed processed mRNA only includes genes that are expressed (Exon)
Hybridization
Joining complementary base pairs (DNA - DNA or DNA - RNA)
Polymerase Chain Reaction (PCR)
Automated. Primers flank gene of interest, denatures and copies
DNA sequence can be created from a very small sample of hybridization
Agarose Gel
Electrophoresis separates DNA by size (longer = slower)
Dideoxyribonucleotide (ddNTP)
Modified base with a 3’H (lack a 3’OH) that causes sequencing of DNA to cease
Gene Therapy
In diseases where gene is mutated or inactive, a normal copy of the gene is inserted into the affected tissue
Transgenic Mice
Gene introduced into germ line (gametes)
Knockout Mice
Created by deleting genes of interest
Chimera
Contain cells from two different lineages
Southern Blotting
Detect the presence and quantity of various DNA strands in a sample. After electrophoresis the sample is transferred to a membrane that can be probed with single stranded DNA molecules to look for the sequence of interest
