BIMM 100 UCSD

Phenol

Removes protein in heat killed bacteria

UV light

Removes DNA & RNA in heat killed bacteria

Adenine

Guanine

Uracil

Thymine

Cytosine

Only fraction of DNA transcribed & some RNA accumulate at much higher levels than others

Why ratio of bases in RNA different than in DNA

DNA polymerase needs

Primer, dNTP, DNA template

Primase, DNA Polymerase, Nuclease, DNA Polymerase, DNA Ligase

Enzymes in replication

Telomeres

A compound structure at the end of chromosomes consisting of 1000's of sequence repeats

Reverse transcriptase

What type of enzyme is Telomerase?

Origin of replication, 2 telomeres & centromere

What is needed to make an artificial chromosome?

DNA polymerase, 3' to 5' exonuclease activity

Proofreading by which enzyme and in what direction?

Exonuclease

Type of activity that cleaves the nucleotides at the ends of the DNA molecule

Endonuclease

Type of activity that cleaves the nucleotides in the middle of the DNA molecule

Mismatch excision repair

Type of repair for incorrect nucleotide pairing not proofread, after replication

Base excision repair

Type of repair for chemically changed bases (deamination), after replication

Nucleotide excision repair

Type of repair for distorted DNA (UV irradiation) after replication

Bypass synthesis

When repair machinery doesn't know what to do

1. Break recognized & exonucleases remove NTPs leaving 3' end ssDNA overhangs

2. ssDNA searches for homologous sequence in sister chromatids

3. Replication 3' end of broken DNA as primer & sister DNA as template

4. Repaired strand base pairs with other broken strand

5. Gap filled by DNA polymerase & ligase

Homologous recombination steps

After S phase to early G2 phase

When is Homologous recombination possible?

1. Recognition of broken ends

2. Exonucleases remove overhang NTPs

3. Ligation of broken ends

End joining steps

RNA

Primary gene product

1. DNA synthesis

2. Denature

3. Gel electrophoresis

Sequencing using fluorescently labeled ddNTPs

Solitary genes

Genes found only once in genome

Duplicated genes

Multiple genes of close but usually not identical sequences

Alters protein expression levels

How does mutation in the UTR affect the protein product of a gene

Intron

What does bacteria DNA not have

2 primers, DNA template, DNA polymerase, dNTPs

Requirements for PCR

DNA Transposons

A transposition mechanism involving the DNA segment to be cut from the donor DNA and pasted into target DNA

Retrotransposons

A transposition mechanism involving the DNA segment to be transcribed (copied) from the donor DNA into RNA, reverse transcribed back into DNA, and pasted into target DNA

Transposases

What does DNA transposons encode for?

Reverse transcriptases

What does retrotransposons encode for?

1. Retrotransposon transcribed to RNA

2. RNA translated to RT

3. RT reverse transcribes RNA to DNA

4. Synthesis of 2nd DNA strand (retrotransposon)

5. Insertion of retrotransposon to DNA

General mechanism of retrotransposition

Intron > UTR > Coding region

Ratio of genetic material in human genome

Gene cloning

Method of studying gene in separation from remainder genome

Gene inactivation

Method of studying cellular/organismal effect of loss of gene function

Selectable marker gene & origin of replication

What is required in a plasmid to ensure bacteria retain it through multiple generations?

Add primers to genomic DNA, PCR product, ligase into vector, transform bacteria, select

Creating a genomic DNA insert using PCR

Add primer to single mRNA, reverse transcriptase into DNA with dNTPs, RNaseH, add primer to ss-cDNA, PCR product to ds-cDNA, ligase into vector, transform bacteria, select

Creating cDNA from mRNA

Each colony has bacteria containing same plasmid, different colonies have different plasmid

Describe colonies of bacteria transformed with genomic DNA

1. Generate DNA with selectable marker & regions flanking the gene to be knocked out

2. Recombine DNA into genome by homologous recombination

Gene inactivation in yeast

1. Create knock-out ES cells by homologous recombination

2. Inject recombinant ES cells into blastocyst, transfer to pseudo-pregnant mouse

3. Cross with wild-type, screen for heterozygous

4. Mate heterozygotic mice

Gene inactivation in mice

CRISPR-Cas9

System that allows specific cleavage in genome directed by complimentary crRNA

Template strand

3' to 5' strand of DNA during transcription

Coding strand

5' to 3' strand of DNA during transcription

1. Isolate RNA

2. Denature & separate RNA by electrophoresis

3. Transfer to membrane & hybridize with radiolabeled probe

4. Detect using autoradiography

Northern blotting steps

1. Isolate total RNA

2. Convert to DNA using reverse transcriptase

3. PCR for cDNA & monitor accumulation

4. More RNA input = faster PCR product accumulation

Quantitative RT-PCR monitor steps