Genetics Exam 3

Benefits of using PCR

1. Not limited by the distribution of restriction enzyme cleavage sites.
2. No host cells are involved—it is an in vitro reaction
3. Requires very little starting material because DNA is amplified

Limitation of PCR

Requires knowledge of the DNA sequence in the region of interest. In other words, primers are necessary.

PCR Primers

Short DNA molecules which delimit the DNA sequence that will be amplified. There is a left one (5’ to 3’) and a right one (3’ to 5’), one for each strand.

Steps of PCR

1. Denature (heat up to 95C)
2. Anneal primers (-55C)—primers, dNTPs and DNA polymerase are added.
3. Extend primers (72C)—essentially DNA replication. A thermal stable polymerase is used (taq polymerase) because it is not denatured at high temperatures.

taq polymerase

The DNA polymerase used in PCR amplification, because it is not denatured by the high temperatures required by PCR.

Southern Blot

Used to detect and quantify DNA. DNA is digested with EcoRI restriction enzyme, transferred to a membrane, and then hybridized with a radioactive probe and run on gel.

Northern Blot

Used to detect and quantify RNA. It must be denatured on a special gel because of its complex structure. Then, like in southern blot, it is transferred to a membrane and hybridized with the probe.

Western blot

Used to identify and quantify proteins. First, proteins are run on a gel, to separate by size. Then they are transferred to a membrane and finally they are incubated with a probe. In this method, antibodies instead of a genetic sequence are used as the probe.

What is the purpose of the membrane in southern/northern/western blot methods?

To make the molecules (DNA, RNA or proteins) more accessible—gel is dense and the molecules need to sit on top of the (usually paper) membrane.

What is the radioactive probe in southern/northern blot methods?

A known sequence that is hybridized with the DNA/RNA. It is the sequence of interest that is being looked for. It is usually labeled. This sequence is able to bind even if it is partially complementary.

What is the probe in western blots

an antibody

Which methods can be used to analyze/quantify DNA?

southern blot, PCR, and high throughput sequencing

Which methods can be used to analyze/quantify RNA?

northern blot, RT-PCR, and high throughput sequencing

Which methods can be used to analyze/quantify proteins?

Western blot

RT-PCR (Reverse transcriptase PCR)

It is used to analyze RNA transcripts.

qPCR

Used to quantify the number of transcripts made. Uses a florescent dye to determine how much DNA is present in the reaction.

High throughput sequencing benefit

Can generate a large number of sequences

Limitations of high throughput sequencing

Sequences are mostly anonymous, and only short reads are generated.

High throughput sequencing steps

1. DNA extraction
2. Library generation
3. Align each short sequence to the reference sequence (these are not always perfect matches).

RNA-seq

Quantification of RNA using high throughput sequencing

Steps of RNA-seq

1. Extract RNA
2. Fragment into pieces
3. Generate library
4. Sequence
5. Map to the genome
6. Count numbers of reads
7. Know expression levels of ALL genes.

ChIP-seq

A type of high throughput sequencing used to detect protein-DNA interactions such as chromatin structure, chromatin modifications, and transcription regulation.

ChIP-seq Steps

1. Extract chromatin
2. Fragment into short pieces
3. Pull down using an antibody
4. Purify DNA
5. Perform sequencing
6. Find all genes bound by a protein

Metagenomics

Another high throughput sequencing method used to identify what species are present in a sample.

Metagenomics steps

1. Take an environmental sample
2. Purify DNA
3. Generate library
4. Sequence
5. Identify all species present in the sample

Possible applications of high throughput sequencing

* find a mutation causing a certain phenotype
* find mutations associated with disease
* determine the best targeted treatment for a specific cancer
* sequence a new interesting genome

**RNA Polymerase**

catalyzes the addition of each ribonucleotide to the 3’ end of the nascent strand. This forms a phosphodiester bond between the 5’ carbon of one nucleotide and the 3’ carbon of the adjacent nucleotide

**Messenger RNA (mRNA)**

Used to encode the sequence of amino acids in a polypeptide. May be polycistronic (encoding two or more polypeptides) in bacteria and archaea. Encode single polypeptides in nearly all eukaryotes

**Ribosomal RNA (rRNA)**

Along with numerous proteins, helps form the large and small ribosomal subunits that unit for translation of mRNA

**Transfer RNA (tRNA)**

Carries amino acids to ribosomes and binds there to mRNA by complementary base pairing to add the amino acids to the elongating polypeptide

**Small Nuclear RNA (snRNA)**

Found in eukaryotic nuclei, where multiple snRNAs join with numerous proteins to form spliceosomes that remove introns from precursor mRNA

**Micro RNA (miRNA)**

Eukaryotic regulatory RNAs that function by base pairing with certain mRNAs, altering their stability and efficiency of translation

**Small Interfering RNA (siRNA)**

Eukaryotic regulatory RNAs that function by base pairing with certain mRNAs, altering their stability and efficiency of translation

**Telomerase RNA**

Located in the telomerase ribonucleoprotein complex, where it acts as a template to maintain and elongate telomere length of eukaryotic chromosomes.

The four stages of transcription

1. Promoter recognition and identification
2. The initiation of transcript synthesis
3. Transcript elongation
4. Transcription termination

**Coding strand/Nontemplete strand**

The complementary strand 

**Promoter**

Region upstream of the start of transcription. It regulates transcription, but is not transcribed

**Coding Region**

The portion of the gene that is transcribed into mRNA

**Termination Region (transcription)**

The portion of the gene that regulates the cessation of transcription

**Bacterial RNA Polymerase core**

Composed of five polypeptide subunits, αI and αII, two β subunits, and an ω subunit

**Holoenzyme**

An active compound composed of multiple subunits. Describes the complete active RNA polymerase, composed of the five polypeptide subunits and the sigma subunit

**σ subunit**

Induces a conformational change in the core enzyme that switches it to its active form. It must be present for the RNA polymerase to bind to the promoter region and initiate transcription (only in bacteria)

**RNA Polymerase**

Transcribes the DNA template strand sequence into RNA.

**Alternative sigma subunits**

impart conformational changes to the core that changes the specificity for the promoter region, enabling transcription of specific genes

**Pribnow box sequence/-10 consensus sequence**

In the promoter region, upstream from the start of transcription, and has the sequence 5’- TATAAT - 3’. It is separated by about 25 bp from the **-35 consensus sequence:** identified by the nucleotide sequence 5’-TTGACA-3’.

**-35 consensus sequence**

consensus in the promoter region identified by the nucleotide sequence 5’-TTGACA-3’.

**+1 nucleotide**

1. The holoenzyme moves downstream and starts transcription at the **+1 nucleotide**

**Closed Promoter Complex (bacterial transcription)**

When the holoenzyme binds to the double stranded promoter sequence

**Open promoter complex (bacterial transcription)**

The holoenzyme, bound to the promoter region, unwinds 18bp of DNA around the -10 consensus sequence

**Intrinsic termination**

Termination occurs as a consequence of termination sequences encoded in the DNA (prokaryotes)

**Inverted repeat sequences**

termination sequences that fold into a complementary loop

**hairpin structure**

In termination, the DNA turns into a hairpin structure—a stem and loop formation.

**Rho-Dependent Termination**

Less common than intrinsic termination, and requires the action of the rho-protein

**RNA polymerase I (eukaryotes)**

Responsible for transcribing several rRNA genes

**RNA polymerase II (eukaryotes)**

Responsible for transcription of mRNAs, and small nuclear RNA genes

**RNA polymerase III (eukaryotes)**

Transcribes all tRNA genes, one small nuclear RNA gene, and one rRNA gene

**TATA box/ Goldberg-Hogness box**

One of three consensus sequences in the promoter region (in eukaryotes), it is at about -25 bp and has the sequence TATAAA. Most frequently present

**CAAT box**

 Located near -80bp when it is present

**GC-rich box**

Has the sequence GGCGG located near -90.

Number of RNA polymerases in prokaryotes

One

Number of RNA polymerases in eukaryotes

Five

**Transcription factors (TF)**

bind to the promoter region, and interact with RNA polymerase to initiate transcription

**TFII**

Transcription factors that influence mRNA transcription by interacting with RNA polymerase II.

Protein that binds to the TATA box. This forms the **initial committed complex**

Contains six proteins and RNA polymerase.

**Preinitiation Complex (PIC)**

Contains six proteins and RNA polymerase

**General Transcription Factors (GTFs)**

Aid in the initiation of transcription, and fall off of RNA polymerase once transcription begins

**Enhancer Sequences**

 Increase the transcription of DNA by binding specific proteins, which interact with the proteins bound at promoter regions. Together, these regions drive the transcription of certain genes

**Splicer Sequences**

DNA elements that act to repress transcription of target genes

**Regulation of gene transcription by chromatin**

Chromatin can change its structure to allow or block RNA polymerase from binding to promoter regions and transcribing certain DNA

**Pre-mRNA**

mRNA before post-transcriptional processing

**Mature mRNA**

mRNA after post-transcriptional processing

**Modification Steps in Posttranscriptional processing**

1. 5’ capping
2. 3’ polyadenylation
3. Intron splicing

**5’ capping**

The addition of a modified nucleotide to the 5- end of mRNA

**3’ polyadenylation**

cleavage of the 3’ end of mRNA and addition of a tail of multiple

 **poly-A tail**

The long chain of adenine nucleotides that is added to a messenger RNA during post-transcriptional processing.

**Intron splicing**

RNA splicing to remove introns and ligate exons

**Functions of the 5’ cap**

1. Protecting mRNA from rapid degradation
2. Facilitating mRNA transport across the nuclear membrane.
3. Facilitating subsequent intron splicing
4. Enhancing translation efficiency by orienting the ribosome on the mRNA.

**Functions of the poly-A tail**

1. Facilitating transport of mature mRNA across the nuclear membrane.
2. Protecting mRNA from degradation
3. Enhancing translation by enabling ribosomal recognition of messenger mRNA.

**RNase**

An enzyme that destroys RNA

**Torpedo model**

After polyadenylation, there is still a residual segment attached to RNA pol II. RNase destroys this segment, and then catches up with RNA pol II, and causes it to dissociate from the strand, terminating transcription

**Four Groups of Introns**

1. Group I (self-splicing, in both eukaryotes and prokaryotes)
2. Group II (self-splicing, in both eukaryotes and prokaryotes)
3. Pre-mRNA (spliceosome, only in eukaryotes)
4. rRNA and tRNA (enzymatic splicing, in both eukaryotes and prokaryotes)

**Pre-mRNA splicing**

When introns are removed after transcription and before posttranscriptional processing by the spliceosome

**Branch site**

Consensus sequence located upstream of the 3’ end of the intron. Contains the branch point adenine

**Spliceosome**

Acts like a “workbench” to which pre-mRNA is attached. The subunits of the spliceosome cut and splice the introns in a four step process. The five ribonucleoproteins that make up the spliceosome are called snRPS

**Alternative pre-mRNA splicing**

A pre-mRNA can be spliced in alternative patterns so that the same transcript might produce different forms of mature mRNA (and thus different proteins)

**Alternative promoters**

Initiate transcription at different +1 start points

**Alternative polyadenylation**

Uses different polyadenylation signal sequences in a gene to produce different mRNAs

**Self-splicing**

**Group I** introns are large catalytically active RNAs (ribozymes) that can catalyze their own excision from mRNA, tRNA and rRNA precursors in bacteria and eukaryotes.

**Group II** introns are found in bacteria, and in organellar DNA in eukaryotes.

**RNA editing**

is responsible for post-transcriptional substitutions of some of the nucleotides of an mRNA

**Guide RNA**

gRNA edits genes in eukaryotes

Purines

Adenine and guanine

Pyrimidines

Cytosine and thymine (uracil in RNA)

**Base Pair Substitution Mutations**

Substitution of one nucleotide base pair by another

**Transition Mutations**

One purine replaces another (A, G), or one pyrimidine replaces another (C, G)

**Transversion Mutations**

A purine is replaced by a pyrimidine or vice versa

**Synonymous (silent) Mutation**

Change in the mRNA codon does not change the amino acid it codes for

**Missense Mutation**

Results in an amino acid change

**Nonsense Mutation**

Creates a stop codon

**Frameshift Mutations**

An addition or deletion of one or more base pairs alters the reading frame, and changes the amino acid sequence from the point of the mutation to the end of the polypeptide chain

**Regulatory Mutations**

Occur in noncoding regions of genes such as promoters, introns and UTR regions. These regions do not encode for amino acids but mutations in these regions can create abnormal or different amounts of mRNAs, which produce mutant phenotypes