Life Sci (Robinson)

What are the parts of molecular cloning?

DNA Preparation, Ligation, Transformation

restriction site

• has multiple DNA sequences recognized by enzymes

• enzymes cut these sequences to insert DNA into the chosen plasmid

promotor sequence

DNA sequence controls rate of transcription

antibiotic resistance gene (bacterial selection)

• cloned plasmids require this

• confirms which bacteria have taken up space

selectable marker

• not an ARG

• grows mammalian cells, kills cells without selectable plasmid

• e.g neomycin

choosing a restriction site

• choose restriction sequence present in restriction site but absent in the sequence of interest to prevent cutting sequence early

Kaiso DNA Sequence

• zinc finger transcription factor, binds to specific DNA sequences

• GGATCC

• involved in Wnt signaling (pathway for cellular differentiation and development)

Kaiso DNA in humans

• through tissue biopsy, DNA isolation, polymerase chain reaction (PCR) testing

• cultured cells derived from human tissues, PCR texting

• someone already cloned Kaiso into an expression construct, cut it out of the old plasmid and put into another plasmid

cDNA

• complimentary DNA through reverse transcription

• no introns

Why are protein coding genes transcribed into mRNA?

• make oligo-DT primer

• allows for reverse transcriptase to dock onto, reads it backwards to create double stranded complimentary DNA

oligo-DT primer

binds to poly A tail of mRNA to initiate cDNA synthesis

relationship between complimentary DNA sequence and genomic DNA in base pairs

Complimentary DNA sequence is shorter than in base pairs

elements of genetically engineered plasmids?

restriction sites, promoter sequence, origin of replication, antibiotic resistance gene, selectable marker

mRNA

RNA without the introns

molecular cloning workflow

select plasmid, target DNA isolation, create recombinant DNA, propagate recombinant DNA in bacteria/host, screen and select bacteria that express DNA, isolate recombinant DNA for further verification

2 enzymes to cut plasmid

• Less chance of the primers reattaching and recirculating because they recognize each other

• We want the Kaiso sequence to be recognized instead

heat shocking

creates pores in the bacterial membrane to force plasmids into bacteria

differentiate molecular cloning and organismal cloning

• molecular cloning is to create many copies of recombinant DNA using a host organism

• organismal cloning is making copies of an entire organism

recombinant DNA

an engineered DNA molecular that comprises a target DNA sequence of interest that has been molecularly cloned into a bacterial plasmid

DNA preparation

• fragment + vector with matching restriction sites

• vector + vector with matching restriction sites

restriction enzymes cut at restriction sites

ligation

• digested fragment (cut into smaller pieces)/ digested vector + vector

• attached by DNA ligase, produces assembled DNA (circle)

exceptions to the central dogma of molecular genetics

rRNA and tRNA

key steps in central dogma of molecular genetics?

Pervasive transcription to produce functional RNA molecules

What are the purines?

guanine, adenine

What are the pyrimidines?

uracil, thymine, cytosine

Differentiate the functional structure of ribonucleic and deoxyribonucleic acid.

• RNA consists of a sugar and nitrogenous base with two hydroxyl groups

• DNA consists of a sugar and nitrogenous base with one less hydroxyl group (on carbon 2)

Differentiate nucleosides and nucleotides.

nucleosides lack a phosphate group, nucleotides contain phosphate group

What happens if a nucleotide is missing a hydroxyl group?

it cannot form bonds

phosphodiester bonds

• significant in DNA and RNA

• forms long nucleotide chains

How else do DNA and RNA structures differ?

DNA is usually double-stranded, RNA is single stranded. DNA does not fold in on itself, RNA can because its structure maintains through phosphodiester bonds. DNA uses thymine, RNA uses uracil.

chromatin

DNA wrapped around positively charged histones, forming compact chromosomes.

base pairing

• occurs through hydrogen bonds

• adenine and thymine form 2 bonds

• guanine and cytosine form 3 hydrogen bonds

C value paradox

discrepancy between genome size and organismal complexity

G value paradox

discrepancy between the number of protein-coding genes and genome size

How does splicing contribute to the C-value and G-value paradoxes?

non-coding regions and alternative splicing; mRNA coding genes contain exons needed for coding, and introns are spliced out

regulatory parts of the genome

promotor, enhancer, silencer

distal promotors

• can be enhancers or silencers depending on enhancing or slowing

• Very far upstream / downstream from transcription start sites

terminator sequences

RNA polymerase stops transcription upon reading these

mutations

Changes in the DNA sequence

differentiate germline and somatic mutations

germlines are heritable, somatic are non-heritable

types of mutations

• single/point mutations

• chromosomal rearrangements

• changes in chromosome number

nonsense mutation

point mutation, results in the code for a stop codon, truncates the protein

missense mutation

point mutation, codes for a different amino acid, results in a non-functioning or different functioning protein

silent mutation

point mutation, no functional change to the protein

How does alternative splicing contribute to protein diversity?

allows a single gene to code for multiple proteins through different splicing mechanisms, like constitutive splicing, mutually exclusive splicing, exon skipping, etc

Key steps in the expression of protein-coding genes?

Transcription, Splicing, Translation, Post-translational Modifications

transcription

DNA is converted into mRNA

translation

ribosomes assemble amino acids into a polypeptide chain

How do spontaneous mutations occur?

• errors during DNA replication

• chemical changes to DNA bases

• environmental factors like radiation

How do spontaneous mutations lead to disease in humans?

• altering protein function

• disrupting gene regulation

• loss of function mutations

• e.g mutation in tumor suppressor genes leading to cancer

mutations are (relative to diversity)

an important source of genetic diversity

spontaneous mutations

• occur randomly with no known cause

• relatively infrequent compared to induced

induced mutations

• exposure to physical and chemical mutagenic agents (radiation)

• mutagenic agents increase mutation rates several fold

effects of mutation on gene function

loss of function, gain of function

loss of function mutation effects

• null (amorphic): complete loss of protein function

• hypomorphic: reduced activity due to less protein produced

• dominant or recessive

haplo-insufficient genes

loss of function in which one normal allele is not enough for normal function

gain of function mutation effects

• increased activity or new function

• hypermorphic: more protein or efficient protein

• neomorphic: generates novel function

• dominant negative or anti-morphic: prevents the normal protein from performing its homeostatic function

• almost always dominant

tautomer

a transient isomeric form of a nitrogenous base

how do point mutations happen

incorporation of a tautomer during DNA replication can result in a single base pair substitution

indels

frame shift mutations, changes the polypeptide sequence of the protein

how do small indels happen?

replication slippage or omission of a nucleotide

how do large indels happen

several hundred to thousands of nucleotides, could be unequal crossing over of chromosomes during meiosis

trinucleotide repeats

3 nucleotides that are repeated several times in succession

e.g CAG CAG CAG CAG (a few to ~50 times)

• prone to mutation

expanded trinucleotide repeat

insertion that arises due to replication slippage in regions of the genome with trinucleotide repeats