Answers in Studying Molecular Biology

How does human genetic information differ from our closest relative?

FOXP2 that encodes for a speech/language development protein split from the ape linage 6 million years ago

Are humans genetically programmed to contract certain diseases?

Answers in Studying Molecular Biology

Cystic fibrosis with an occurrence of 1 in 3,000 for U.S. babies is caused by a defective protein resulting in high lung secretions

Healthy Lung

Cystic Fibrosis LungCystic Fibrosis Lung

DNA: The Genetic Code

Deoxyribonucleic acid (DNA) was identified in 1944 as the information required to transform normal bacterial strains to pathogenic ones

In 1951 DNA and not proteins was confirmed as the infectious agent for bacteriophages (viruses that infect bacteria)

DNA: The Genetic Code

Chargaff’s rule (A+G = C+ T)

Watson and Crick DNA structure elucidation (1953)

Bases that Constitute DNA

At what position does the purine or pyrimidine base link with the ribose sugar backbone?

Nucleotides (NTPs) versus Deoxy-nucleotides (dNTPs)

RNA ribose sugar

DNA 2’-deoxyribose sugar

What is the difference between a nucleotide and a nucleoside?

DNA Double Helix Stability

Base π stacking and

Base pair H-bonding

DNA Structural Features

Anti-parallel polynucleotide strands

Right-handed helix

Sugar-phosphate exterior exposed to solvent

Mg+2 stabilizes phosphate groups

Two unequal grooves

Axial View of DNA

Sugar-phosphate exterior exposed to solvent

DNA Melting Curve

UV absorption greater with double- versus single-stranded DNA

DNA can re-nature with single strands re-forming a double helix if cooling occurs slowly.

DNA Re- and De-naturation

In nature, DNA can be unwound by helicases and cut by endo- or exo-nucleases at phosphodiester bonds

DNA Melting Point Depends on GC Content

RNA versus DNA Components

Sugar hydroxylation pattern

Base methylation pattern

RNA Structural Features

Less stable

Normally single stranded

Can fold and base pair with complementary segments of the same strand

Transfer RNA molecule

Other Nucleotides Molecules/Functions

Acetyl unit transferase

+ R-C(O)CH3

Other Nucleotides Molecules/FunctionsOxidation/Reduction Reactions

Other Nucleotides Molecules/FunctionsOxidation/Reduction Reactions

Central Dogma of Molecular Biology

DNA, RNA and proteins, are linear polymers (i.e. each monomer is connected to at most two other monomers)

The monomer sequence effectively encodes information

DNA Replication

Proteins catalyzed:

[i] unwinding of the super helix – DNA helicases

[ii] unwinding of the double helix – DNA helicases

[iii] replicate the master template – DNA polymerases

DNA Transcription

RNA polymerase and transcription factors catalyze messenger RNA (mRNA) formation from DNA sections

In eukaryotes the primary transcript (pre-mRNA) is processed via alternative splicing

Nucleotides (NTPs) versus Deoxy-nucleotides (dNTPs)

RNA ribose sugar

DNA 2’-deoxyribose sugar

What is the difference between a nucleotide and a nucleoside?

RNA to Protein Conversion: Translation

Mature mRNAs attach to ribosomes where it is read as a triplet codon

Initiation and elongation factors bring aminoacylated transfer RNAs (tRNAs) into the ribosome-mRNA complex

Standard Amino Acid Genetic Code

Cystic Fibrosis Protein Function/ Gene Structure

Diagnostic signs of CF include:

High chloride concentration in sweat

Thick mucus in the airways

A Mutation Site Resulting in Cystic Fibrosis

Sanger or Dideoxy DNA Sequencing

Components for the Chain Termination Method:

• DNA fragment for sequencing

• Primers

• dNTPs

• ddNTPs

Sanger or Dideoxy DNA Sequencing

What direction does the DNA polymerase elongate the DNA fragment?

Sanger or Dideoxy DNA Sequencing Migration smaller is faster through the gel (bottom smallest)

Each peak represents 1 base

Pyrosequencing Based on DNA synthesis

Template DNA is immobilized

Peak area represents base number

http://www.pyrosequencing.com/DynPage.aspx?id=7454

Gene Amplification by Polymerase Chain Reaction (PCR)Gene specific amplification without purification

Amplification via temperature cycling

Special Taq (Thermus aquaticus) DNA polymerase

Detection of Helicobacter pylori, Borrelia burgdorferi, as well as hepatitis, HIV, and West Nile virus

Restriction Enzyme Recognition Sites

Blunt and Sticky DNA Cuts

Restrictive endonucleases recognize 4-8 bp sequences within the DNA and cleave at a specific site

DNA strands that are cut in a symmetric fashion are palindromic

Molecular Cloning by Recombinant DNA Technology• Cut by restriction enzymes

• Anneal an uncatalyzed reaction

• Re-close by DNA ligase

Screening for Colonies Containing the Plasmid with the Insert

Size-Specific Cloning Vectors

Recombinant Protein Products

Generating a Restriction Map

Where are the restriction sites located on the 20 kb fragment that would generate this map?

.

.

.

Transcript Analysis via Microarray/DNA Chip

mRNA↓

cDNA fluorecent labeling↓

cDNA/oligonucleotideHybridization

↓Slide scanning

↓Data analysis

How to examine gene expression changes with a given treatment

Differential Arabidopsis Gene Expression with GB03 Exposure

Full Image

Block 8

RNA Extraction

cDNA Synthesis

Label

Hybridize

Wash

Scan

HN

O

NO

OH

O

O

PO

O-

O

NH2

O

PO

O-

O

P

O-

-O

NN

SO3-

O

ON

O

O

-O3S

cDNA Synthesis and Labeling

Amino Allyl-dUTP

Cy5 Dye

Microarray Data Analysis

Chapter 3 Problems:

1, 2, 7, 8, 9, 13, 15, 19, 21, 25, 29, 61, 62,