2. DNA and Molecular Tools

four characteristics of genetic material

1. must be able to replicate

2. must store information

3. must express information

4. must allow variation by mutation

how is genetic material transmitted from parent to offspring?

physically transmitted

what component was favored as genetic material for a long time, before DNA was confirmed as the genetic material?

protein

evidence favoring DNA as the genetic material

• Griffiths transformation experiment

• Avery, MacLeod and McCarty’s experiment

Griffiths transformation experiment

showed a “transforming principle” transforming a non-pathogenic bacteria into a pathogenic bacteria by the introduction of a dead pathogenic bacteria with the non-pathogenic bacteria
living pathogenic bacteria was recovered from the deceased mice used in the experiment

Avery, MacLeod and McCarty’s experiment

showed that the transforming principle was DNA, not protein

nucleotides

building blocks of DNA

a nucleoside with a phosphate group added

nucleoside

a nitrogenous base and the pentose sugar

components of a nucleotide

• a nitrogenous base

• a pentose sugar

• a phosphate group

where is the location of the phosphate group on a nucleotide?

C5

where is the location of the phosphodiester bond on a nucleotide?

C5 phosphate - C3 OH

pentose sugar in DNA

deoxyribose

pentose sugar in RNA

ribose

nitrogenous bases in DNA

A, T, C, G

nitrogenous bases in RNA

A, U, C, G

nucleoside monophosphate

another term for nucleotides

how many phosphate groups can a nucleotide have?

one (DMPs), two (NDPs) or three (NTPs)

polynucleotides

nucleotides linked together by phosphodiester bonds

Watson-Crick model of DNA

• right-handed double helix

• two strands are antiparalell

• base stacking

base pairing on DNA

A-T

G-C

how many base pairs per helix turn in DNA?

10

how are antiparallel trans held together

hydrogen bonds

how many hydrogen bonds in A-T?

23’

how many hydrogen bonds in G-C?

3

5’ end of DNA

free phosphate

3’ end of DNA

free OH

mode of DNA replication

semiconservative

semiconservative replication

complementarity of DNA allows each strand to serve as a template for synthesis of the other

Meselson-Stahl experiment

demonstrated semiconservative DNA replication in prokaryotes

how are genes passed from parents to offspring?

from gametes through meiosis

types of viruses

can have RNA or DNA

DNA virus example

T4 bacteriophage

RNA virus example

HIV retrovirus

retroviruses

• replicates in an unusual way through reverse transcription

• DNA produced can be incorporated into the host cells genome

reverse transcription

uses reverse transcriptase to turn RNA into DNA, in retroviruses this allows the DNA to integrate into the host cells genome

central dogma of biology

transcription → translation → protein

nontemplate strand of DNA

coding strand

the result of transcribing the template strand

template strand of DNA

noncoding strand

degenerative

multiple codons exist for a single amino acid

codon

3 nucleotides

two amino acids specified by only a single codon

tryptophan and methionine

frame shift mutation

insertion of bases to genetic code not in multiples of three, disrupting all subsequent codons

recombinant DNA

cloning to amplify or express DNA

methods for detection of nucleic acids

southern blot and northern blot

methods for detection of proteins

western blot

how to clone a piece of DNA into a vector

1. enzyme digestion to generate DNA fragments

2. DNA ligase cuts fragments

3. introduce ligated DNA into host cell

4. amplify DNA and isolate recombinants

three essential features of vectors for DNA cloning

1. number of convenient restriction sites

2. marker gene to select for presence in host cell

3. origin of replication to multiply plasmid in host cells

restriction enzymes

cleave DNA at a specific sequence

sticky end ligation

two DNA strands cut unevenly on each strand pair complementarily on the single strand overhang, and are joined by DNA ligase

how to amplify a specific gene in vitro without cloning/host cell

polymerase chain reaction (PCR)

three steps of PCR

1. denaturation

2. primer annealing

3. extension

repeat using a thermocycler

materials for PCR

• DNA template

• primer

• taq DNA polymerase

• dNTP

taq DNA polymerase

a thermostable DNA polymerase (will not denature in thermocycler)

what does a southern blot detect?

DNA

what does a northern blot detect?

RNA

dideoxy chain termination sequencing

most common method of DNA sequencing

dideoxynucleoside

has no 3’ OH to form phosphodiester bond, terminates transcription

steps for dideoxy chain termination

1. primer is bound to template

2. add DNA polymerase, dNTPs and small amount of ddNTPs

3. primer is extended, terminated when ddNTP is added

4. strands are separated by size using gel electrophoresis and analyzed automatically for sequencing

southern blot

detects a DNA fragment in a mixture of DNA fragments using a radioactively labeled single-strand probe that is a compliment to the gene of interest
the probe is detected using an x-ray

northern blot

similar to a southern blot, but instead of DNA, RNA is separated on a formaldehyde gel

uses of northern blot

• to detect if a gene is being expressed in a tissue

• determines the size of the mRNA/coding region

microarrays

detects RNA expression in bulk

how to make lots of proteins by expressing foreign genes in E. coli

1. clone gene of interest to E. coli expression vector

2. transform recombinant vector to E. coli

3. induce protein expression in E. coli

4. purify protein from E. coli

western blot analysis

separates proteins by molecular weight in a gel, then transferred to nitrocellulose
the blot is probed with an antibody to a specific protein, a secondary antibody is used to visualize the band (radioactivity or fluorescence)

immunofluorescence to detect protein localization in cells

• primary antibodies binds proteins in cells

• fluorescent secondary antibodies bind to primary antibodies

three ways to determine gene function

1. cell culture

2. generation of transgenic animals

3. RNAi

how to determine gene function in a cell culture

express gene in a cell culture to study its effect on the cell

how to determine gene function in transgenic animals

observing loss or gain of function mutations

production of transgenic animals via microinjection

microinject plasmid DNA into pronucleus of fertilized egg, then transfer the embryo to a foster mother

RNAi

degrades target mRNA or inhibits expression of target mRNA

transfection

introduce DNA into tissue culture cells

how retroviruses are used as vectors to express a gene in a cell

recombinant retroviruses carrying the gene of interest are reverse transcribed and integrated into the hosts genome. the gene of interest is later transcribed and expressed in cells

mutation via homologous recomination

a cloned, mutated copy of a gene is inserted into the cells and recombined with the normal chromosomal copy. after homologous recombination, the cell is carrying the mutated gene in place of the normal copy