2.8 Polymerase Chain Reactions (PCR) and Transcription & Translation

What is PCR?

Polymerase Chain Reactions are used to make millions of copies of DNA for scientists to study

What are 4 things we need for PCR?

1. DNA Sample

2. Taq Polymerase

3. Free DNA Nucleotides

4. Manufactured Primers for specific sequences (STRs)

What is the importance of Taq Polymerase in the process of PCR?

Since it is heat resistant, its used to protect primers from denaturing during the process of PCR.

What machine are PCR “ingredients” put into to begin the process of PCR?

A Thermocycler

What are the 4 stages/temperatures that the thermocycler goes through in one cycle?

1. Room Temperature

2. 95C, DNA Denaturing

3. 50C, Primer Annealing

4. 70C, Primer Extension

What feature is used to determine if a DNA sample is good for DNA profiling? How do we do this?

Multiple Short Tandem Repeats (STRs) from a DNA sample are counted to see if the number matches the original DNA sample.

What is a non-coding sequence?

Sequences that do not code for the formation of a polypeptide

What are 4 examples of non-coding sequences?

1. STRs

2. Enhancers: turn genes on

3. Silencers: turn genes off

4. Telomeres: repetitive sequences that cap ends of chromosomes for protection

What is a Variable Number Tandem Repeat (VNTR)?

The collective number of repeats for multiple STRs

Which technique is used to analyze STRs?

Gel Electrophoresis, which doesn’t tell us exactly what bases there are, but does tell us the size and charge of STRs.

How can we tell the size of STR molecules through gel electrophoresis?

STR samples will travel further and faster from their starting point if they are smaller because they have less mass, so they can be pulled through the gel more easily.

What measurement is used to describe fragment length in Gel Electrophoresis?

number of base pairs (bp)

What happens during Transcription?

RNA Polymerase unzips DNA to begin translation, which creates mRNA

How is mRNA made?

1. Transcription begins at a Promoter: a DNA sequence at the beginning of a gene

2. RNA Polymerase binds to the DNA Template Strand and transcribes from 5’ to 3’

3. Transcription stops when a Terminator Sequence is reached

What are 3 modifications made to mRNA to make it usable?

1. Poly-A Tail: Adds stability and prevents degradation on the 3’ end of the mRNA

2. Methyl Cap: Adds protection and export guidance (methyl is chemically attracted to nuclear pores) on the 5’ end of the mRNA

3. Splicing

Explain the process of the mRNA Splicing modification.

A spliceosome (mRNA combined with snRNPs) cleave the mRNA to join exons and remove introns to increase genetic diversity in samples

What happens during Translation?

tRNA and mRNA are brought together by ribosomes to assemble polypeptides

What are the steps of Translation?

1. Small Ribosomal Subunit (SU) binds to the initiator tRNA (usually Met), forming the initiation complex

2. SU binds to 5’ end of mRNA

3. Initiation complex moves along mRNA until it reaches the start codon (usually AUG)

4. tRNA links to start codon by hydrogen bonding

5. Large Ribosomal Subunit binds to the intiation complex w/tRNA in the P site

6. Next codon is now in the A site and translation begins

What happens after Translation?

1. Proteins are modified before being functional (Methyl/Prosthetic Groups)

2. Proteins fold into secondary/tertiary/quaternary structure

3. Unused/old proteins are broken down by large proteins called proteasomes into amino acids that can be recycled to make new proteins

What are the 3 sites on the mRNA where tRNA operates on?

Exit Site, Peptide Site, Accepting Site

What occurs at start/stop codons?

Ribosomes are disassembled

How do nucleic acids function as genetic code? What hypothesis did this lead to?

A certain sequence of codons is what determines the sequence of amino acids in a polypeptide.

The “one gene one polypeptide” hypothesis: each gene is responsible for making one polypeptide

What is a Polysome? Why is this useful?

A group of ribosomes that are bonded to the mRNA to translate the same mRNA strand. This allows us to create the same number of identical proteins as there are ribosomes.

What are 3 types of Gene Expression Regulation?

1. Transcriptional: Mechanisms that prevent/encourage transcription

2. Post-Transcriptional: Control/regulate mRNA after its produced (ex. Splicing)

3. Translational: Mechanisms that increase translation

What are two aspects of DNA that may decrease Transcription?

1. Methylation: When more methyl groups are added to nucleosomes/histones/directly on top of the DNA, making it harder for polymerase to bind to it

2. Nucleosome Spacing: When there is less space between nucleosomes, making it harder for polymerase to read it

Which amino acid is usually attached to the initiator tRNA?

Methionine (AUG)