Pre-7.01 Getting up to Speed in Biology
- Source:: Getting up to Speed in Biology | MIT Open Learning Library
- Instructor:: Hazel Sive, Diviya Ray
- Offered by:: MIT OpenCourseWare
- Platform:: MIT OpenLearningLibrary
- Publish Date:: 2019
- Review Date::
About this Course
This self-paced course was originally designed to help prepare incoming MIT students for their first Introductory Biology course (known at MIT as 7.01). It will also be useful for anyone preparing to take an equivalent college-level introductory biology class elsewhere.
# Lecture 1 - Molecules of Life
# Lecture 1.1 - Representing Molecules
- Life is made of cells. ^408e50
- building block
- small (human ~10uM diameter)
- made of large (macro)molecules
- full of chemical reactions
- Bonds build molecules. ^d68145
- holds molecules together
- boding is due to attraction between atoms
- In life, there are some key elements that come up again and again. ^37e186
- Carbon — C
- Oxygen — O
- Nitrogen — N
- Phosphorous — P
- Sulpher — S
- valency = number of bonds an atom can make ^d86973
- Representing molecules
# Representing Molecules
# Chemical Formula
Each C, H, and O atom is counted. For example…
# Full Structure
All atoms and bonds are shown. For example…
# Line-angle Structure
Below is butane. Carbons are at the corners or ends, and hydrogens are not shown.
The locations of the carbons are shown below with blue dots.
Now, the locations of the hydrogens are shown below with red dots.
Because C has a valency of FOUR (meaning that each C makes four bonds), you can figure out location of H atoms.
# Lecture 1.2 - Polar and Non-polar Molecules
- Electronegativity (en) is the attraction of an atom for electrons ^595807
- Polar molecule ^9468b7
- Unequal en across the atoms
- Non-polar molecules ^ce8892
- equal en across atoms
- Polarity depends on ^f2a6fd
- polar bonds where the electrons are unequally distributed (dipole)
- geometry of the molecule
An unequal electron distribution is called a dipole.
Dipoles are often shown by a T-arrow or a δ. The dipoles are shown on the figure in red. The overall dipoles (which are a vector sum of the individual dipoles) are shown in blue.
# For CO2…
The C=O bond is POLAR since O is more electronegative than C (attracts electrons more), BUT the vector sum of the dipoles is equal to zero, so CO2 is NON-POLAR overall.
# For H2O…
The O-H bond is POLAR since O is more electronegative than H. The vector sum of dipoles is NOT equal to zero, so H2O is POLAR overall.
# Example: Propane v. Propanol
This is propane.
Chemical formula: C3H8
Bonds: NON-POLAR, since C and H share electrons equally
This is propanol.
Chemical formula: C3H7OH or C3H8O
Bonds: C-O and O-H bonds are POLAR, since O is more electronegative (attracts electrons more) than C or H
Molecule: POLAR, the dipole vectors do not cancel out.
# Lecture 1.3 - Types of Bonds
- Hydrogen (big in bio)
- Van der Waals/non-polar (hydrophobic)
# Ionic Bond
An ionic bond is an electrostatic interaction between oppositely charged ions (atoms that have lost/gained 1 or more electrons).
This image is of acetate CC(=O)[O-] and of a sodium ion [Na+]. The sodium ion is positioned next to the negatively-charged O atom of the acetate. The negatively charged oxygen and the positively charged sodium are circled to indicate that they interact via an ionic bond.
# Covalent Bond
A covalent bond consists of one or more pairs of electrons are shared by two atoms.
This image is of acetate CC(=O)[O-] and of a sodium ion [Na+]. The sodium ion is positioned next to the negatively-charged O atom of the acetate. Double bond between the central carbon and the neutral charge oxygen is circled to indicate that the interaction is a type of covalent bond.
# Hydrogen Bond
A hydrogen bond forms between polar molecules where an H has a partial positive charge (induced by an electronegative atom) and is therefore attracted to a different electronegative atom.
# Non-polar/van Der Waals Bond
This type of bond is between nonpolar molecules, with transient unequal charge distribution (dipoles).
# Lecture 1.4 - Recognizing Macromolecules
- There are 4 major classes of macromolecules in cell
- They are often polymers, i.e. some combination of monomers. (monomer = M, polymer = $M^n$).
- Lipids ^cbd454
- Cell membranes, Insulation, Signaling, Store energy
- Always or largely non-polar (hydrophobic) — key charachteristic
- or amphipathic (partly polar)
- Long chain or small
- Carbohydrates ^20ebf7
- Blood type, energy, information, exoskeleton, plant cell wall, DNA
- Can be recognized by their basic chemical formula $CH_2O$.
- Monomer can usually be identified: it is monosaccharides (sugars)
- Monomers will form long chains to create polymers (glycogen/starch)
- M joined by glycosidic bond C-O-C
- Nucleic Acids ^17e881
- They make genes.
- Made up of nucleotide (M), polymers are either DNA or RNA.
- Structure is comprised of phosphate — sugar — base: P-S-B ^757a96
- sugar = ribose (RNA) or deoxyribose (DNA)
- 4 bases
- A, G (purines)
- C, T (pyrimidines)
- A, G, C, T = DNA
- A, G, C, U = RNA
- They do everything, except carry the hereditary information.
- Made up of monomer = amino acid.
- 20 common amino acids.
- Protein is an amino acid polymer.
- The structure is built around alpha carbon.
- alpha carbon + NH2 + COOH + R = side group
- R can be polar, non-polar, charged, uncharged.
- Can be written in three ways, i.e. full, three-letter, or one-letter.
- Recognizing macromolecules
Lipids are non-polar and hydrophobic.
Carbohydrates follow the formula CH2O and are polar.
Proteins are made up of amino acid monomers, which consist of an α-carbon (circled in red) bound to an NH2 group, a COOH group, and a side chain, or R group, (circled in blue). The R group can be polar or non-polar
# Nucleic Acids
Nucleic acids are built from nucleotide monomers, which consist of a phosphate group (circled in green), a sugar (circled in orange), and a base (circled in purple). Nucleotides are polar. ^86dbdb
# Lecture 1.5 - Nucleic Acid Polarity
- Macromolecules carry information, because they have different ends and directions.
- Macromolecules may have order and polarity = information.
- Nucleic Acid Polymer
- 5’P — S — P — S — P — S3’
- Always write 5’ and 3’ ends when writing, ALWAYS
# Anatomy of a Nucleic Acid
Above is a diagram of a nucleic acid polymer.
Recall that each nucleotide is composed of a sugar, phosphate and base.
S = Sugar
P = Phosphate
The strand is polymerized from the 5’ end to the 3’ end. The nucleotide at the 5’ end has a free phosphate, and the nucleotide at the 3’ end has a free 3’OH group on its sugar.
S-P backbone is not written, just the bases and the polarity of the strand.
ALWAYS write 5’ and 3’ on each nucleic acid strand!
In other words, written nucleic acids take the following form:
# Polarity of Synthesis
Nucleic acids are made from 5’P to 3’OH. This means that the –3’OH end is the growing end, i.e. receives the incoming nucleotide, as shown below.
Base order = INFORMATION
Polarity = 5’ and 3’ ends show first to last nucleotide added and confer a direction to read information
# Lecture 1.6 - Protein Polarity
- peptide bond between NH_2 and COOH.
- polarity due to
- amino (N), carboxy (C) end
- information = amino acid order
- C = last amino acid added
- ALWAYS write N and C on each protein.
# Anatomy of an Amino Acid Polymer
The diagram above shows the synthesis of a dipeptide from two amino acid monomers.
The alpha-carbons have been circled in orange.
The peptide bond that forms upon synthesis is circled in blue.
The polypeptide is polymerized from the N end to the C end. The amino acid at the N end has a free NH2 group and the amino acid at the C end has a free COOH group.
Proteins are written with three or one letter amino acid code from N to C.
ALWAYS write N and C on each protein!
OR, from the diagram above:
# Polarity of Synthesis
A polypeptide chain is synthesized from N to C. Therefore, the –C end is the growing end (for addition of next amino acid).
Amino acid order = INFORMATION
Polarity = N and C ends show first to last amino acid added
# Lecture 2 - The Cell and how it Works
# Lecture 2.1 - Condensation and Hydrolysis
- Cellular Chemistry
- Metabolism = all chemical reactions in a cell
- Two types of chemical reactions
- break down
- build up
- they release water as they react
- Think of cell as a factory
# Anabolism v. Catabolism
Anabolism is a form of metabolism that consumes energy and by which covalent bonds are formed.
Catabolism is a form of metabolism that produces energy and by which covalent bonds are broken.
# Condensation Reactions
A condensation reaction is one that forms covalent bonds and produces water as a product. These reactions are anabolic.
A-OH + H-B → A-B + H2O
(This reaction is also included in the figure at end.)
# Hydrolysis Reactions
Hydrolysis reactions break covalent bonds by consuming a water molecule and dividing its atoms between separate molecules. These reactions are catabolic.
A-B + H2O → A-OH + H-B
(This reaction is also included in the figure below.)
# Lecture 2.2 - Free Energy and Reaction Kinetics
- All chemical reactions are governed by “free energy” (G).
- Here, free really means “usable”.
\Delta G = \Delta H - T \Delta S
- There are three cases that may occur during a reaction:
- $\Delta G$ is negative
- Energy release, reaction proceeds
- $\Delta G$ is positive
- requries energy to proceed
- $\Delta G$ is zero
- equilibrium, where R —> P = P —> R, i.e. reactants become product at the same rate as the products go back and become reactants.
- $\Delta G$ is negative
- Catalyst are a class of chemicals that decrease EnAC (activation energy) to promote reaction.
- Enzymes are biological catalysts, mostly protein.
- They are specific for each reaction.
- They don’t change delta G but increase rate of reaction.
# Free Energy (ΔG)
ΔG = ΔH - TΔS
H = Enthalpy
S = Entropy
# Exergonic Reactions
Exergonic reactions release energy, so that ΔG < 0 and the reaction is spontaneous.
# Endergonic Reactions
Endergonic reactions require energy to proceed, so that ΔG > 0 and the reaction is non-spontaneous.
If ΔG = 0, the reaction is at equilibrium such that the rate fo the forward reaction = the rate of the backwards reaction.
# Lecture 2.3 - Pathways
- Enzyme reactions organized into pathways
- multistep pathways
- feedback control
- positive (make more) and negative (make less) feedback loops
- internal/external signals control pathways
# Biochemical Pathways
Biochemical pathways include activation (positive control), represented by a pointed arrow (→),
and inhibition (negative control), represented by a T-bar arrow.
Feedback serves to regulate product levels within a pathway.
# Lecture 2.4 - Organelles
- Cells are the building blocks of life.
- The cell is surrounded by plasma membrane/cell membrane.
- Membrane is made of amphipathic lipids, and they form a bilayer.
- It is largely hydrophobic. So, cell is effectively sealed in a kind of plastic bag.
- Various channels/pores allow polar molecules through the cell membrane.
- Cells are replicating, membrane-bound factories.
- Organelles are the subcellular structures with specific function inside the cell.
- They may be membrane-bound.
# What is a Cell?
A cell is the smallest unit of life that is bound by a membrane and can self-replicate.
There are two types of cells: eukaryotic cells (such as the cells that make up plants and animals) and prokaryotic cells (such as bacteria cells).
An organism that is made up of one or more eukaryotic cells is called a eukaryote, and an organism whose cells are prokaryotic is called a prokaryote. In this way, plants and animals can be referred to as eukaryotes and bacteria are examples of prokaryotes.
An organelle is defined as a specialized structure within a cell that serves a specific function.
The chart below lists eukaryotic organelles with a short summary of their functions. Pay particular note to the nucleus, mitochondrion (plural: mitochondria), and ribosome.
|Common Organelles of Eukaryotic Cells|
|Organelles with membranes|
|Nucleus||Protecting, controlling access to DNA|
|Endoplasmic Reticulum (ER)||Routing, modifying new polypeptide chains; synthesizing lipids; other tasks|
|Golgi body||Modifying polypeptide chains; sorting, shipping proteins and lipids|
|Vesicle||Transporting, storing, or digesting substances in the cell, other functions|
|Mitochondrion||Making ATP by breaking down sugars|
|Chloroplast||Making sugars in plants and some protists|
|Peroxisome||Inactivation of toxins|
|Organelles without membranes|
|Ribosome||Assembling polypeptide chains|
|Centriole||Anchor for the cytoskeleton|
# Lecture 2.5 - Cell Division
- Cells make more cells.
- It is one of the main attributes of life.
- Cell division cycle has mainly two parts:
- DNA replication
- It makes two sets of the genes.
- DNA partitioning
- It divides the DNA between daughter cells.
- DNA replication
- Genes are organized in chromosomes (chr).
- Body cells contain 2 of each chr.
- It is called diploid, also written as 2n.
- The matching chromosomes are called homologs/homologous.
- Germ cells (egg/sperm) contain one of each chr.
- It is called haploid, also written as n.
- There are two kinds of cell division:
- in body (somatic) cells
- Thousands or millions of cells in a human body are undergoing mitosis at any particular time.
- Outcome of this is 2 cells (daughter cells) that are identical to the parent cell (2n).
- DNA (chrs) replicates.
- chrs line up on ‘spindle’ (=microtubules).
- one copy of each chr is partitioned to the daughter cell.
- cell membrane partitions daughter cells.
- parent, daughter cells all DIPLOID = 2n each.
- produces germ cells (egg/sperm)
- outcome is 4 cells, not identical to parent, each is Haploid, n = 1 copy of each chr
- DNA (chr) replication
- Then chr pair, may exchange DNA segments.
- Two steps to Meiosis
- Meiosis 1
- Each replicated homologous chr pair goes to daughter cell.
- Outcome is two cells.
- Meiosis 2
- Cells divide again.
- A single homolog goes to each daughter cell.
- Meiosis 1
Mitosis is the process of cell division by which 2 daughter cells are produced from 1 mother cell.
The daughter cells have the same ploidy and are genetically identical to both each other and the mother cell.
Meiosis is the process of cell division by which 4 gametes are produced from 1 mother cell.
The gametes’ ploidies are half of the mother cell’s ploidy. The gametes are NOT genetically identical to each other NOR to the mother cell.
Gametes are cells with half of the normal ploidy (haploid for humans). Examples of gametes are eggs (ova) and sperm cells.
# Lecture 3 - Information Transfer in Biology
# Lecture 3.1 - DNA Rules
- What is a gene?
- A unit of hereditary.
- Nucleic acid instructions for product
- Usually DNA
- Molecular biology includes biological information transfer.
- Gene (DNA) replicates, i.e. is copied into RNA via Transcription, which is then translated into protein.
- DNA (RNA) Rules
- Base pairing, A = T, C =- G
- complementary DNA strands
- antiparallel strands, 5’ to 3’ and 3’ to 5’
# DNA Polarity
Recall that DNA strands have a 3’ end and a 5’ end and that new nucleotides are added to the free -OH group of the 3’ end.
# Base Pairing
For DNA, there are 4 nucleotide bases: Guanine (G), Cytosine (C), Adenine (A), and Thymine (T).
G pairs with C between complimentary strands via the formation of 3 hydrogen bonds.
Similarly, A pairs with T via the formation of 2 hydrogen bonds.
For RNA, the bases are the same, except Uracil (U) is used instead of Thymine.
In RNA, A pairs with U, and they form 2 hydrogen bonds.
(Therefore, the RNA bases are: G, C, A, U.)
# Lecture 3.2 - DNA Replication
- DNA replication produces DNA from a DNA template.
- process takes place within nucleus of the cell.
# The Steps of DNA Replication
Step 1. The two strands of the original DNA separate.
Step 2. RNA primers attach to the separated DNA strands.
Step 3. The DNA is replicated off of the 3’ end of the RNA primers.
Thus, the result of DNA replication is two semiconservative, identical DNA molecules.
# Lecture 3.3 - Transcription
- Transcription produces RNA that is copied from a DNA template.
- Difference b/w replication and transcription
- RNA has U vs DNA has T
- Only one DNA template strand is used.
An RNA molecule is synthesized from one strand of a dsDNA; this strand used for synthesis is called the template strand.
Like DNA, RNA is synthesized 5’ to 3'.
Once finished, the RNA transcript dissociates from the template strand, and the two DNA strands hybridize again to form dsDNA.
# Lecture 3.4 - Translation
- Translation is a process that produces protein from mRNA template.
- Genetic code refers to triplets of RNA bases (called codon), each of which encodes one amino acid.
- Requires tRNA (transfer RNA), ribosomes.
- Uses mRNA (messenger RNA) as template
- RNA is read from 5’ to 3’ prime
- amino acids add to C end
- SO protein is made N –> C
- tRNA = Interpreter
- Recognizes codon
- A tRNA ‘anticodon’ and mRNA ‘codon’ base pair
- tRNAs also carry correct amino acids
Translation is the process by which RNA is decoded by the ribosome to produce a polypeptide.
A codon is a set of 3 consecutive nucleotides that together code for an amino acid.
Adaptor RNA molecules called tRNAs bring amino acids to the ribosome.
The tRNAs have anticodons that pair (antiparallelly) with the codon to ensure that the correct amino acid is being added.
# The Codon Chart
The codon chart is used to determine which amino acid corresponds to which codon. Notice that some amino acids have multiple codons but each codon only codes for one amino acid.
Here is a codon chart.
When using this chart, remember that nucleic acids are read from 5’ to 3’. Therefore, the first nucleotide, or letter, will always be the one closest to the 5’ end and the third nucleotide will always be the one closest to the 3’ end.
# Lecture 4 - Inheritance and Genetics
# Lecture 4.1 - Genes to Proteins
- DNA (gene) –> RNA –> Protein —> ‘Trait’
- Trait = observable characteristic = ‘phenotype’
- DNA base changes = mutations
- may alter protein sequence/function, therefore, may change a trait
- Two types
- Point mutation — Change 1 base at a time
- Point mutation — Change 1 base at a time
- Mutations can change ‘control’ DNA —> alter RNA synthesis and protein levels
# Types of Mutations
A point mutation is a change in a single nucleotide base.
Point mutations may result in…
- a missense mutation, in which the changed nucleotide base results in a single amino acid change in the protein product.
- a silent mutation, in which the changed nucleotide base does not result in any changes to the amino acids in the protein product.
- a nonsense mutation, in which the codon that includes changed nucleotdie base changes from coding for an amino acid to a stop codon that terminates translation.
An insertion is the addition of an extra nucleotide(s) within the sequence. Similarly, a deletion is the elimination of a nucleotide(s) from the sequence.
Insertions and deltions frequently result in frameshift mutations, by which the extra or missing nucleotide bases change the reading frame (the grouping of three adjacent nucleotides into codons), thus resulting in a change in the amino acids that are encoded by that nucleotide sequence.
# Lecture 4.2 - Allele Segregation
- An allele is an alternate form of a gene.
- May be relative to ‘wild type’
- An allele is due to DNA sequence difference (variation)
- In diploid cells, each chromosomes pair has same or different alleles of a gene.
- Alleles encode slightly different proteins.
An allele is a version of a gene that determines which trait will be expressed. This occurs because each allele encodes slightly different versions of the same protein product.
# Segregation of Alleles
In mitosis, each daughter cell will end up with the same alleles as the original mother cell.
Therefore, if the mother cell possesses alleles B and b for gene 1, then both daughters will have both B AND b.
However, in meiosis, each gamete produced will have only one allele of each gene from the mother cell.
Therefore, if the mother cell possesses alleles D and d for gene 2, then each gamete will have either D OR d.
Which allele a gamete will inherit for one gene is independent of which allele it will inherit for another gene. This means that all of the allelic combinations shown in the diagram below are equally likely to arise in the gametes from the mother cell.
# Lecture 4.3 - Punnett Squares
- Genetics: tools to understand gene function/inheritance
- Monohybrid cross = 1 trait (gene)
- homozygous = BB
- heterozygous = Bb
- Genetic cross follow traits
# A List of Genetic Terminology
- Genotype = genes of the individual
- Phenotype = observable characteristics
- Gene = a unit of hereditary (inheritance)*
- Allele = alternate forms of a gene; for example, the gene “apple” could have different forms (different DNA sequences) such as “appleB” and “appleb”
- Gametes = germ cells, egg or sperm
- Generation = individuals born at the same time from the same parents or their parents’ siblings
- P = parent
- F1 = first generation offspring
- F2 = second generation
*NOTE: this is the genetic definition of a gene. In terms of molecular biology, a gene is defined as the DNA instructions for a product.
# Dominant v. Recessive
When in combination, different traits can be either dominant or recessive. If a trait is dominant, it will be expressed whenever an allele for that trait is present. If a trait is recessive, it will be expressed ONLY when the allele for that trait is present AND dominant alleles are absent.
For example, for a gene determines color, there may be an allele (A) that codes for red color and an allele (a) that codes for green color.
If A is dominant to a (meaning that a is recessive to A), then AA = red, Aa = red, and aa = green.
However, some traits have more complicated expression patterns. With the same example gene, incomplete dominance would result in a new, blended trait when different alleles are present.
For example: Aa = orange.
There is also codominance in which the traits of both alleles are expressed, leading to a mixed trait.
For example: Aa = red and green spots.
# Punnett Squares
Punnett Squares are a method to visually represent the likelihood of potential genotypes (and their resulting phenotypes) of the progeny of a cross (the F1 generation) based on the genotypes of the parents.
# Lecture 4.4 - Pedigrees
- Pedigree = genetics from family history
- circle = female
- square = male
- filled = trait present
- Affected children born to TWO unaffected parents = recessive trait
- Every affected child has an affected parent = dominant trait
- Two types of chromosomes + pedigrees
- autosomes = paired
- sex chrs:
- XX female - paired
- XY male - unpaired
- autosomal recessive — males and females affected equally
- autosomal dominant — males and females affected equally
- X-linked recessive — males affected more
- genes on X: transmitted to SONS via mother
# Pedigrees and Modes of Inheritance
A pedigree is a visual diagram used to map out the inheritance of genetic traits within a family. Pedigrees are often useful for determining the mode of inheritance, or inheritance pattern, of a particular trait.
Genetic traits can display various modes of inheritance. They can be either dominant or recessive, and they can be either autosomal or X-linked. This course focuses on the following modes of inheritance:
- Autosomal recessive: a recessive trait that is inherited on any chromosome that is not X or Y
Pedigrees of autosomal recessive traits exhibit affected offspring from TWO unaffected parents (both sexes should be affected equally).
- Autosomal dominant: a dominant trait that is inherited on any chromosome that is not X or Y
For pedigrees of autosomal dominant traits, every affected offspring has one affected parent (both sexes should be affected equally).
- X-linked recessive: a recessive trait that is inherited on the X chromosome (this means that females will each have two alleles and males will only have one)
Pedigrees of X-linked recessive traits will often have only males affected.
# Pedigree Nomenclature
# Lecture 5 - Building with DNA
# Lecture 5.1 - Restriction Digests
- Genetic Engineering
- a.k.a. Recombinant DNA Technology
- means DNA construction made in lab
- Molecular cloning
- Making a lot of the same DNA = clone
- What is the process of genetic engineering?
- Basic steps of building a Recombinant DNA
- Gene of Interest (GOI)
- Cut out GOI DNA from larger piece
- Paste GOI into ‘vector’ (DNA)
- Vector = carrier of extra DNA that replicates
- Grow lots of clone DNA
- Basic steps of building a Recombinant DNA
- Cut DNA
- via Restriction Endonucleases (RE)
- enzymes that recognize, bind, and cut specific DNA sequences
- Two types of ends of cut DNA
# Restriction Endonucleases
A restriction endonuclease, sometimes referred to simply as a restriction enzyme, cuts double-stranded DNA at palindrome sequences. Each restriction endonuclease cuts at one specific sequence. After being cut, the double-stranded DNA is left with “sticky ends”. Different restriction endonucleases leave different types of sticky ends, which are reviewed below.
# Types of Sticky Ends
Sma1 leaves blunt ends:
EcoR1 leaves sticky (5’ overhang) ends:
PstI leaves sticky (3’ overhang) ends:
# Lecture 5.2 - Compatible Ends
- Vector = virus that grows in bacteria
- Often circular = plasmid
- has ‘ORI’ = origin of DNA replication = start site of DNA synthesis
- allows vectors to replicate to $10^4$ copies per cell —> Lots of DNA
- GOI is ‘pasted’ into vector via DNA ligase
- enzyme that joins compatible (matching) DNA ends
- any two blunt ends can ligate
- two complementary sticky ends can ligate
- mar or may not reform a restriction site
- Molecules with ‘ase’ at the end are usually enzymes.
# Big Picture: Cloning
Cloning is the process of isolating a gene of interest (GOI) and expressing it in an organism.
A vector is any carrier of DNA that can deliver that DNA, i.e. the GOI, to the cell and be replicated. One common example of a vector is a plasmid, which is a piece of small, circular DNA.
The cloning process is depicted in detail below.
# Restriction Endonucleases: Compatible Ends
Restriction endonucleases, discussed in the previous section, are used to cut out and isolate the GOI (step 3 in the diagram above) and to cut the vector so that the GOI can be integrated into the vector (step 4 in the diagram above).
In order for the GOI to integrate into the vector, the two need to be cut in a way that gives them compatible ends. In this way, they can match up and ligate into a new circular plasmid without any breaks. The different types of compatible ends are detailed below.
Note that the GOI and the vector do not need to be cut with the same enzyme to be compatible. In the case of blunt ends, all blunt ends can match with other blunt ends. In the case of sticky ends, only the overhanging base pairs need to match up.
However, ligating DNAs from two different restriction sites may prevent the ligation point from serving as a restriction site in the future because you alter the sequence on the other half of the restriction site (in the case of blunt ends) or around the overhanging base pairs (in the case of sticky ends).
# Lecture 5.3 - PCR
- Exponential amplification (synthesis) of DNA in lab
- dsDNA = double-stranded DNA
- Primer = short peice of complementary DNA
# The PCR Process
The following diagram depicts the process of PCR (Polymerase Chain Reaction), which has 3 main steps:
- primer annealing
- extension (synthesis of new DNA by the DNA polymerase, Taq Polymerase)
It is important to note that this reaction, while based off of the principle of DNA replication, happens in a test tube (not a cell!) with the goal of amplifying a DNA sequence.
# Notes about PCR Primers
The primers used in this reaction are DNA primers, not RNA primers, because they are synthetically made and added to the test tube. The primers should anneal to the outermost region of the sequence that you would like to amplify. For each reaction, you should have two types of primers: one to extend along the top strand of the DNA sequence and one to extend along the bottom strand.